Anti-ror1 antibodies

ABSTRACT

The invention relates to antibodies, and in particular, to antibodies exhibiting specificity for Receptor tyrosine kinase-like Orphan Receptors (ROR), and to uses thereof, for example in the treatment of cancer. The invention extends to polynucleotide and polypeptide sequences encoding the antibodies, and therapeutic uses thereof, and to diagnostic kits comprising these molecules. The invention also extends to antibody-drug conjugates and to uses thereof in therapy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 62/193,237, filed Jul. 16, 2015, and U.S.Provisional Application No. 62/163,241, filed May 18, 2015, each ofwhich is incorporated by reference herein in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is FIVE_004_02WO_ST25.txt. The text file is 185 KB,was created on May 17, 2016, and is being submitted electronically viaEFS-Web, concurrent with the filing of the specification.

FIELD OF THE INVENTION

The present invention relates to antibodies, and in particular, toantibodies exhibiting specificity for Receptor tyrosine kinase-likeOrphan Receptors (ROR), and to use thereof, for example in the treatmentof cancer. The invention extends to polynucleotide and polypeptidesequences encoding the antibodies, and compositions comprising thesemolecules and therapeutic uses thereof. Diagnostic kits and methodscomprising these molecules are also described. The invention alsoextends to antibody-drug conjugates and to uses thereof in therapy.

BACKGROUND OF THE INVENTION

Receptor tyrosine kinase-like orphan receptors (ROR) belong to aconserved family of receptor tyrosine kinases, which consists of twofamily members, ROR1 and ROR2, which are type-I transmembrane receptortyrosine kinases. The extracellular region of ROR1 and ROR2 contains animmunoglobulin (Ig) domain, a cysteine-rich domain (CRD), also called aFrizzled (Fz) domain, and a Kringle (Kr) domain. All three domains areinvolved in protein-protein interactions. Intracellularly, ROR1 and ROR2possess a tyrosine kinase (TK) domain and a proline-rich domain (PRD)straddled by two serine/threonine-rich domains

The cellular function of this family is to regulate cell migration,planar cell polarity (PCP) and apical-basal cell polarity, and axonoutgrowth in developmental processes, including skeletal and neuronaldevelopment. Wnt5a, a glycoprotein critical in carcinogenesis, has beenidentified as regulating these functions by binding and activating ROR1and ROR2 (Nishita et al., 2010, Trends Cell Biol. 20(6):346-54). Wnt5abinding to ROR2 and its co-receptor, Frizzled domain, can activate theINK pathway and filamin A to regulate cell migration and invasion, causeRacl and Rho A to regulate cell polarity, and induce Src family membersto modulate the expression of matrix metalloproteases, such as MMP 1, 2,13, and inhibit the canonical Wnt pathways. Unlike ROR2, the molecularmechanism behind the regulation of ROR1 cellular function is still notclear. A recent study showed that ROR1 could promote cell proliferationthrough NF-B when co-expressed with Wnt5a (Fukuda et al., 2008, ProcNatl. Acad Sci USA. 105(8):3047-52). ROR proteins are embryonicproteins. In mice, they are expressed only during the developmentalstage. ROR expression is quickly silenced after birth, and isundetectable in adult tissues. ROR1 or ROR2 knockout mice exhibitneonatal lethality and die shortly after birth. Mice with ROR1 knockoutdevelop normally and show no visible phenotype. Mice with ROR2 knockoutexpress skeletal defects such as shortened snouts, limbs, tails and acleft palate, and a defect in the membranous part of ventricular septum.

ROR1 is aberrantly expressed in B-cell chronic lymphocytic leukemia(CLL) and mantle cell lymphoma (MCL). Like its mouse counterpart, humanROR1 expression cannot be detected in normal blood cells and other adulttissues, apart from low levels in adipose tissue. Studies havedemonstrated that knockdown of ROR1 and fibromodulin results insignificantly increased apoptosis of CLL cells (Choudhury et al., 2010,Br J Haematol. 151(4):327-35).

CLL is the most common form of human leukemia in the Western hemisphere.According to the Leukemia and Lymphoma Society, approximately 16,000 newcases of CLL are diagnosed in the U.S. each year, and 4,400 people diefrom the disease annually. CLL is characterized by the accumulation offunctionally immature cells in the bone marrow, blood, lymph tissue andother organs. It is a malignancy of mature B-cells, and most oftenaffects adults over the age of 55, of which two-thirds are men, thoughsometimes occurs in young adults. There is a significant unmet medicalneed for new and improved therapeutic options for CLL. Despite treatmentusing chemotherapy and stem cell transplantation, CLL is an incurabledisease with average five year survival of only 50%.

It has been reported, with statistical significance, that a high levelof ROR1 and ROR2 expression is correlated with a lower survival rate inneuroblastoma patients (FIGS. 1 and 2). Gene expression profiling datafrom pre-B ALL patients also suggest a correlation between ROR1upregulation and t(1; 19) translocation, which generates the E2A-PBX1fusion protein and serves as a biomarker of a subtype of pre-B-ALL (FIG.3).

Recent advances in monoclonal antibodies (mAbs) for oncology indicationshave yielded new options for the treatment of CLL, either as stand-alonetherapies or in combination with chemotherapy regimens. In 2007, Campath(anti-CD52 humanized antibody) received FDA approval as a first-linetreatment for CLL. Previously, Campath was approved as a second-linetreatment for CLL patients that stopped responding to alkylating agentsand Fludara. Campath treatment showed significantly superior patientresponse compared to using chlorambucil as an initial treatment. Campathalso exhibited a favorable toxicity profile. Rituxan, an anti-CD20antibody drug, was approved by the FDA for treatment of patients withCLL in combination with two other chemotherapy drugs, fludarabine andcyclophosphamide. With all of these advances, approximately 20% ofpatients still do not achieve complete disease control, and mostpatients eventually developed resistance to the available therapies.Therefore, there is still a great need for more effective and innovativeCLL therapies.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the invention, and to show how embodimentsof the same may be carried into effect, reference will now be made, byway of example, to the accompanying diagrammatic drawings.

FIG. 1 is a graph showing statistically significant lower survival ofneuroblastoma patients with high levels of ROR1 gene expression.

FIG. 2 is a graph showing statistically significant lower survival ofneuroblastoma patients with high levels of ROR2 gene expression.

FIG. 3 shows ROR1 mRNA expression in pre-B ALL patients.

FIG. 4 represents the ROR1 phage display panning strategy.

FIG. 5 is a graph showing an example of ROR1 phage antibody ELISAresults.

FIG. 6 is a graph showing an example of ROR1 phage antibody cell ELISAresults.

FIG. 7 illustrates methodology for affinity ranking by phage competitionELISA.

FIG. 8 illustrates epitope Class I antibody CDR sequence analysis.

FIG. 9 illustrates epitope Class II antibody CDR sequence analysis.

FIG. 10 illustrates epitope Class III antibody CDR sequence analysis.

FIG. 11 illustrates epitope Class IV antibody CDR sequence analysis.

FIG. 12 is a graph showing an example of the binding specificity of ROR1positive phage clones by ELISA.

FIG. 13 is a graph showing an example of positive phage clones bindingto both human ROR1 and human ROR2 by ELISA.

FIGS. 14a and 14b are plasmid maps of human anti-ROR1 full-length IgG1antibody expression vectors.

FIG. 15 shows protein sequences of human antibody constant domains of anembodiment of the anti-ROR1 full-length IgG1 antibody of the invention.

FIG. 16 illustrates SDS-PAGE of purified human anti-ROR1 full-lengthIgG1 antibodies.

FIGS. 17a and 17b show Kd binding affinity analysis of anti-ROR1full-length IgG1 antibodies by bio-layer interferometry.

FIGS. 18A and 18B represent antibody 601-3-2 epitope mapping ELISA data.The highlighted regions illustrate the 601-3-2 epitope in human ROR1protein sequence.

FIGS. 19A and 19B represent antibody 601-3-12 epitope mapping ELISAdata. The highlighted regions illustrate the 601-3-12 epitope in humanROR1 protein sequence.

FIGS. 20A and 20B represent antibody 601-3-16 epitope mapping ELISAdata. The highlighted regions illustrate the 601-3-16 epitope in humanROR1 protein sequence.

FIG. 21a-21c are graphs showing ELISA results of anti-ROR1 full-lengthIgG1 antibodies against human ROR1, human ROR2, and mouse ROR1.

FIG. 22a-22c are graphs showing binding analysis of anti-ROR1full-length IgG1 antibodies by FACS.

FIG. 23 is a graph showing Clq binding of anti-ROR1 full-length IgG1antibodies.

FIG. 24a-24c are graphs showing ADCC activity of anti-ROR1 full-lengthIgG1 antibodies against MDA-MB-231.

FIG. 25 show blocking of Wnt5a ligand binding by anti-ROR1 full-lengthIgG1 antibodies; and

FIG. 26 is a graph showing ROR1 antibody internalization.

FIG. 27 shows normalized sensosgrams of anti-ROR1 antibodies binding toROR1 extracellular domain monomer and dimer forms.

FIG. 28 shows binding of anti-ROR1 antibodies to CLL cells from patient13 at four different concentrations (10, 2.5, 0.63, 0.16 μg/mL from leftto right for each antibody), with signal correction by subtracting MFIobtained for the same concentration of isotype control antibody.

FIG. 29 shows binding of anti-ROR1 antibodies to CLL cells from patient13 at four different concentrations (10, 2.5, 0.63, 0.16 μg/mL from leftto right for each antibody) with signal correction by subtracting theMFI obtained for the same concentration of isotype control antibody.

DETAILED DESCRIPTION OF THE INVENTION

The inventors focused their investigations on Receptor TyrosineKinase-Like Orphan Receptor 1 (ROR1), as they considered it to be apromising novel cancer target for CLL, as well as other cancers.However, the inventors found that a significant problem involved in thedevelopment of effective ROR1-targeting monoclonal antibody diagnosticsand therapies is that ROR1 has very low expression levels on the surfaceof CLL cancer cells. Indeed, studies have shown that ROR1 expression onCLL cancer cells is estimated to be only few thousand molecules per cell(Baskar et al., 2008, Clin Cancer Res. January 15; 14(2):396-404. Incontrast, most antibody therapies target molecules that arehighly-expressed on cancer cell surfaces. For example, Roche's antibodybreast cancer drug Herceptin targets the Her2 antigen, which has anexpression level estimated at >50,000 copies per cell. Accordingly, aneffective antibody treatment for CLL must be able to identify CLL cancercells despite the incredibly low ROR1 copy number. One way to overcomethe challenge of low copy number of ROR1 on cancer cell surface is toidentify a high affinity antibody that can bind to ROR1 positive cancercells efficiently despite the low density of ROR1.

The objective of the present invention therefore is to develophighly-specific and high-affinity anti-ROR1 monoclonal antibodies thatwill target and kill cancerous cells with minimal side effects to normaltissues. By laborious panning against the human ROR1 extracellulardomain (ECD) protein using a fully-human antibody phage library, theinventors have been able to isolate a highly specific human ROR1specific antibody.

Therefore, according to a first aspect of the invention, there isprovided a human anti-Receptor Tyrosine Kinase-Like Orphan Receptor 1(ROR1) antibody, or a functional fragment thereof.

Advantageously, the inventors have isolated the first humanROR1-immunospecific antibody, and developed a ROR1-targeting antibodytherapy for ROR1-positive cancer types, such as B-cell ChronicLymphocytic Leukemia (CLL), the most common form of human leukemia inthe Western hemisphere, thereby addressing an unmet medical need.Targeting ROR1 by the antibody according to the invention for cancertherapy is based on the important scientific finding that ROR1 isaberrantly expressed in cancer cells, but absent in normal blood cellsand normal adult tissues, and therefore provides a highly specifictherapy targeting only cancerous cells and tumors.

As described in the Examples, several different embodiments of the fullyhuman and unique anti-ROR1 antibody of the invention have been isolated,each of these antibodies being capable of recognizing distinct bindingepitopes on ROR1 protein with surprisingly high affinity.Advantageously, as described in the Examples the antibody of theinvention can: (i) mediate complement-dependent cytotoxicity (CDC), (ii)mediate antibody-dependent cellular cytotoxicity (ADCC) against variousROR1-positive cancer cell lines, (iii) block Wnt5a binding to ROR1,and/or (iv) inhibit Wnt5a induced ROR1 phosphorylation. The inventorsbelieve that this is first time that a fully human anti-ROR1 antibodyhas been isolated, identified and sequenced. The antibody of theinvention therefore presents a highly effective therapeutic agent whenused alone, or as a vehicle that is capable of delivering potentanti-cancer reagents, or as an engineered antibody exhibiting enhancedimmune functions. Additionally, the antibody of the invention may alsobe used as a diagnostic or prognostic tool. Advantageously, based on itsunique expression profile as a foetal antigen expressed only duringembryonic development, and preferential expression profiling in multiplecancers, an effective antibody therapy targeting ROR1 will improve andlengthen life for a broad cancer patient population, and reducelong-term healthcare costs.

The invention extends to both whole antibodies (i.e., immunoglobulins)with immunospecificity for a ROR1 protein, preferably an extracellulardomain thereof, as well as to functional fragments thereof. Suchfragments retain at least one antigen binding region of a correspondingfull-length antibody. The antibody or functional fragment thereof maycomprise a monoclonal or polyclonal antibody or functional fragmentthereof.

The antibody or functional fragment may be monovalent, divalent orpolyvalent. Monovalent antibodies are dimers (HL) comprising a heavy (H)chain associated by a disulphide bridge with a light chain (L). Divalentantibodies are tetramer (H2L2) comprising two dimers associated by atleast one disulphide bridge. Polyvalent antibodies may also be produced,for example by linking multiple dimers. A ROR1 antibody of the presentinvention can be a chimeric antibody, bi-specific antibody,multi-specific antibody, humanized antibody and/or human antibody. Thebasic structure of an antibody molecule consists of two identical lightchains and two identical heavy chains which associate non-covalently andcan be linked by disulphide bonds. Each heavy and light chain containsan amino-terminal variable region of about 110 amino acids, and constantsequences in the remainder of the chain. The variable region includesseveral hypervariable regions, or Complementarity Determining Regions(CDRs), that form the antigen-binding site of the antibody molecule anddetermine its specificity for the antigen, e.g., ROR1 or an epitopethereof. On either side of the CDRs of the heavy and light chains is aframework region, a relatively conserved sequence of amino acids thatanchors and orients the CDRs.

The constant region consists of one of five heavy chain sequences (μ, γ,ζ, α or ε) and one of two light chain sequences (κ or λ). The heavychain constant region sequences determine the isotype of the antibodyand the effector functions of the molecule.

As used herein, the term “human antibody” can mean an antibody, such asa monoclonal antibody, which comprises substantially the same heavy andlight chain CDR amino acid sequences as found in a particular humanantibody exhibiting immunospecificity for ROR1 protein. An amino acidsequence, which is substantially the same as a heavy or light chain CDR,exhibits a considerable amount of sequence identity when compared to areference sequence. Such identity is definitively known or recognizableas representing the amino acid sequence of the particular humanantibody. Substantially the same heavy and light chain CDR amino acidsequence can have, for example, minor modifications or conservativesubstitutions of amino acids. Such a human antibody maintains itsfunction of selectively binding to ROR1 protein. Human antibodiesinclude, but are not limited to, antibodies produced in non-humananimals that comprise human immunoglobulin genes, such as XenoMouse®,and antibodies selected using in vitro methods, such as phage display,wherein the antibody repertoire is based on human immunoglobulinsequences.

The term “human monoclonal antibody” can include a monoclonal antibodywith substantially human CDR amino acid sequences produced, for exampleby recombinant methods such as production by a phage library, bylymphocytes or by hybridoma cells.

The term “humanized antibody” can mean an antibody from a non-humanspecies (e.g., mouse) whose protein sequences have been modified toincrease their similarity to antibodies produced naturally in humans Theterm “humanized antibody” refers to an antibody in which at least oneamino acid in a framework region of a non-human variable region (such asmouse, rat, cynomolgus monkey, chicken, etc.) has been replaced with thecorresponding amino acid from a human variable region. In someembodiments, a humanized antibody comprises at least one human constantregion or fragment thereof. In some embodiments, a humanized antibody isan Fab, an scFv, a (Fab′)₂, etc.

A “chimeric antibody” as used herein refers to an antibody comprising atleast one variable region from a first species (such as mouse, rat,cynomolgus monkey, etc.) and at least one constant region from a secondspecies (such as human, cynomolgus monkey, chicken, etc.). In someembodiments, a chimeric antibody comprises at least one mouse variableregion and at least one human constant region. In some embodiments, achimeric antibody comprises at least one cynomolgus variable region andat least one human constant region. In some embodiments, all of thevariable regions of a chimeric antibody are from a first species and allof the constant regions of the chimeric antibody are from a secondspecies.

A “bispecific antibody” as used herein refers to an antibody comprisinga first arm comprising a heavy chain/light chain combination that bindsa first antigen and a second arm comprising a heavy chain/light chaincombination that binds a second antigen. In some embodiments, one of thearms of a bispecific antibody comprises a heavy chain/light chaincombination that binds ROR1.

The antibody may be a recombinant antibody. The term “recombinant humanantibody” can include a human antibody produced using recombinant DNAtechnology.

The term “antigen binding region” can mean a region of the antibodyhaving specific binding affinity for its target antigen, e.g., the ROR1protein. The binding region may be a hypervariable CDR or a functionalportion thereof. The term “functional portion” of a CDR can mean asequence within the CDR which shows specific affinity for the targetantigen. The functional portion of a CDR may comprise a ligand whichspecifically binds to ROR1 protein.

The term “CDR” can mean a hypervariable region in the heavy and lightvariable chains. There may be one, two, three or more CDRs in each ofthe heavy and light chains of the antibody. Normally, there are at leastthree CDRs on each chain which, when configured together, form theantigen-binding site, i.e., the three-dimensional combining site withwhich the antigen binds or specifically reacts. It has however beenpostulated that there may be four CDRs in the heavy chains of someantibodies.

The definition of CDR also includes overlapping or subsets of amino acidresidues when compared against each other. The exact residue numberswhich encompass a particular CDR, or a functional portion thereof, willvary depending on the sequence and size of the CDR. Those skilled in theart can routinely determine which residues comprise a particular CDRgiven the variable region amino acid sequence of the antibody.

The term “functional fragment” of an antibody can mean a portion of theantibody which retains a functional activity. A functional activity canbe, for example antigen binding activity or specificity (e.g., anantigen-binding fragment). A functional activity can also be, forexample, an effector function provided by an antibody constant region.The term “functional fragment” is also intended to include, for example,fragments produced by protease digestion or reduction of a humanmonoclonal antibody and by recombinant DNA methods known to thoseskilled in the art. Human monoclonal antibody functional fragmentsinclude, for example individual heavy or light chains and fragmentsthereof, such as VL, VH, and Fd; monovalent fragments, such as Fv, Fab,and Fab′; bivalent fragments such as F(ab′)2; single chain Fv (scFv);and Fc fragments.

The term “VL fragment” can mean a fragment of the light chain of a humanmonoclonal antibody which includes all or part of the light chainvariable region, including the CDRs. A VL fragment can further includelight chain constant region sequences.

The term “VH fragment” can means a fragment of the heavy chain of ahuman monoclonal antibody which includes all or part of the heavy chainvariable region, including the CDRs.

The term “Fd fragment” can mean the light chain variable and constantregions coupled to the heavy chain variable and constant regions, i.e.,VL CL and VH CH-1.

The term “Fv fragment” can mean a monovalent antigen-binding fragment ofa human monoclonal antibody, including all or part of the variableregions of the heavy and light chains, and absent of the constantregions of the heavy and light chains. The variable regions of the heavyand light chains include, for example, the CDRs. For example, an Fvfragment includes all or part of the amino terminal variable region ofabout 110 amino acids of both the heavy and light chains.

The term “Fab fragment” means a monovalent antigen-binding fragment of ahuman monoclonal antibody that is larger than an Fv fragment. Forexample, a Fab fragment includes the variable regions, and all or partof the first constant domain of the heavy and light chains. Thus, a Fabfragment additionally includes, for example, amino acid residues fromabout 110 to about 220 of the heavy and light chains.

The term “Fab′ fragment” can means a monovalent antigen-binding fragmentof a human monoclonal antibody that is larger than a Fab fragment. Forexample, a Fab′ fragment includes all of the light chain, all of thevariable region of the heavy chain, and all or part of the first andsecond constant domains of the heavy chain. For example, a Fab′ fragmentcan additionally include some or all of amino acid residues 220 to 330of the heavy chain.

The term “F(ab′)2 fragment” can mean a bivalent antigen-binding fragmentof a human monoclonal antibody. An F(ab′)2 fragment includes, forexample, all or part of the variable regions of two heavy chains-and twolight chains, and can further include all or part of the first constantdomains of two heavy chains and two light chains.

The term “single chain Fv (scFv)” can mean a fusion of the variableregions of the heavy (VH) and light chains (VL) connected with a shortlinker peptide.

One skilled in the art knows that the exact boundaries of a fragment ofa human monoclonal antibody are not important, so long as the fragmentmaintains a functional activity. Using well-known recombinant methods,one skilled in the art can engineer a polynucleotide sequence to expressa functional fragment with any endpoints desired for a particularapplication. A functional fragment of the antibody may comprisefragments with substantially the same heavy and light chain variableregions as the human antibody. Preferably, the functional fragment isROR1-specific.

The functional fragment may include fragments wherein at least one ofthe binding region sequences has substantially the same amino acidsequence as the binding region sequences of the antibody, morepreferably the ROR1-specific human antibody. The functional fragment maycomprise any of the fragments selected from a group consisting of VH,VL, Fd, Fv, Fab, Fab′, scFv, F (ab′)₂ and Fc fragment.

The functional fragment may comprise any one of the antigen bindingregion sequences of the VL, any one of the antigen binding regionsequences of the VH, or a combination of VL and VH antigen bindingregions of a human antibody. The appropriate number and combination ofVH and VL antigen binding region sequences may be determined by thoseskilled in the art depending on the desired affinity and specificity andthe intended use of the functional fragment. Functional fragments ofantibodies may be readily produced and isolated using methods well knownto those skilled in the art. Such methods include, for example,proteolytic methods, recombinant methods and chemical synthesis.Proteolytic methods for the isolation of functional fragments compriseusing human antibodies as a starting material. Enzymes suitable forproteolysis of human immunoglobulins may include, for example, papain,and pepsin. The appropriate enzyme may be readily chosen by one skilledin the art, depending on, for example, whether monovalent or bivalentfragments are required. For example, papain cleavage results in twomonovalent Fab′ fragments that bind antigen and an Fc fragment. Pepsincleavage, for example, results in a bivalent F(ab′) fragment. An F(ab′)2fragment of the invention may be further reduced using, for example, DTTor 2-mercaptoethanol to produce two monovalent Fab′ fragments.

Functional fragments produced by proteolysis may be purified by affinityand column chromatographic procedures. For example, undigestedantibodies and Fc fragments may be removed by binding to protein A.Additionally, functional fragments may be purified by virtue of theircharge and size, using, for example, ion exchange and gel filtrationchromatography. Such methods are well known to those skilled in the art.

The human antibody or functional fragment thereof may be produced byrecombinant methodology. Preferably, one initially isolates apolynucleotide encoding desired regions of the antibody heavy and lightchains. Such regions may include, for example, all or part of thevariable region of the heavy and light chains. Preferably, such regionscan particularly include the antigen binding regions of the heavy andlight chains, preferably the antigen binding sites, most preferably, theCDRs.

The polynucleotide encoding the human antibody or functional fragment ofthe invention may be produced using methods known to those skilled inthe art. The polynucleotide encoding the antibody or a functionalfragment thereof may be directly synthesized by methods ofoligonucleotide synthesis known in the art. Alternatively, smallerfragments may be synthesized and joined to form a larger functionalfragment using recombinant methods known in the art.

As used herein, the term “immunospecificity” can mean the binding regionis capable of immunoreacting with a ROR1 protein, by specificallybinding therewith. The antibody or functional fragment thereof canselectively interact with an antigen (e.g., ROR1 peptide) with anaffinity constant of approximately 10⁻⁵ to 10⁻¹³ M⁻¹, preferably 10⁻⁶ to10⁻⁹ M⁻¹, even more preferably, 10⁻¹⁰ to 10⁻¹² M⁻¹.

A “subject” may be a vertebrate, mammal, or domestic animal. Hence,medicaments according to the invention may be used to treat any mammal,for example livestock (e.g., a horse), pets, or may be used in otherveterinary applications. Most preferably, the subject is a human being.

A “therapeutically effective amount” of the antibody or fragment thereofis any amount which, when administered to a subject, is the amount ofagent that is needed to treat the cancer, or produce the desired effect.

The term “anti-cancer composition” can mean a pharmaceutical formulationused in the therapeutic amelioration, prevention or treatment of cancerin a subject.

A “pharmaceutically acceptable vehicle” as referred to herein, is anyknown compound or combination of known compounds that are known to thoseskilled in the art to be useful in formulating pharmaceuticalcompositions.

As shown in FIG. 17, kinetic binding analysis confirmed specific bindingof the full length IgG1 antibodies to hROR1-ECD, with a Kd in picomolarrange. Thus, preferably the KD of the antibody or fragment thereof forROR1 may be less than 1×10⁻¹⁰, preferably less than 1×10⁻¹¹, morepreferably less than 1×10⁻¹². The antibody or fragment thereof mayexhibit an IC50 for ROR1 of about 10⁻⁷ to 10⁻¹⁰ M⁻¹.

The term “immunoreact” can mean the binding region is capable ofeliciting an immune response upon binding with an ROR1 protein, or anepitope thereof.

The term “epitope” can mean any region of an antigen with ability toelicit, and combine with, a binding region of the antibody or fragmentthereof.

As shown in FIGS. 23 and 24, the antibody or fragment thereof may becapable of mediating killing of ROR1-expressing tumor cells. Killing maybe via Complement-Dependent-Cytotoxicity (CDC) and/or viaAntibody-Dependent Cellular Cytotoxicity (ADCC).

As illustrated in FIG. 25, the antibody or fragment thereof may becapable of blocking binding of Wnt5a to ROR1 protein.

As shown in FIG. 26, the antibody of fragment thereof may be capable ofbeing endocytosed upon binding to ROR1.

The antibody or fragment thereof may be capable of blocking Wnt5a ROR1phosphorylation. The antibody or fragment thereof may be capable ofinhibiting cancer cell proliferation.

The term “ROR1” can refer to family 1 of the receptor tyrosinekinase-like orphan receptors (mRNA: NM_005012.2, protein: NP_005003.2).The DNA sequence encoding one embodiment of human ROR1 is providedherein as SEQ ID NO:1.

The polypeptide sequence of one embodiment of human ROR1 is providedherein as SEQ ID NO:2.

As described in the Examples, the inventors have isolated 45 differentembodiments of the antibody or functional fragment according to thefirst aspect of the invention. The inventors have carried out epitopemapping, and found that embodiments of the antibody or fragment thereofare capable of binding to various different epitopes on the ROR1protein, these epitopes being designated herein as epitope class I-IV.Epitope I may be defined as being residues KNDAPVVQEPRRLSFRSTIYGSR (SEQID NO:237; i.e., amino acids 93-115 of SEQ ID NO:2) and AANCIRIGIPMADPI(SEQ ID NO:238; i.e., amino acids 293-307 of SEQ ID NO:2), epitope IImay be defined as being residues SSTGVLFVKFGPPPTASPG (SEQ ID NO:239;i.e., amino acids 141-159 of SEQ ID NO:2) and SNPMILMRLKLPNCE (SEQ IDNO:240; i.e., amino acids 269-283 of SEQ ID NO:2), and epitope III andepitope IV may be defined as being the rest of the extracellularsequence of SEQ ID NO:2. Thus, the antibody or fragment thereof ispreferably capable of binding to one or more of epitopes I to IV on ROR1protein, or a functional fragment or variant of any one of theseepitopes. It will be appreciated that knowledge of these variousepitopes can be used to generate a novel ROR1-specific antibody.

Hence, in a second aspect, there is provided use of an epitope forgenerating an anti-Receptor Tyrosine Kinase-Like Orphan Receptor 1(ROR1) antibody, or a functional fragment thereof, wherein the epitopeis selected from a group of epitopes consisting ofKNDAPVVQEPRRLSFRSTIYGSR (SEQ ID NO:237; i.e., amino acids 93-115 of SEQID NO:2); AANCIRIGIPMADPI (SEQ ID NO:238; i.e., amino acids 293-307 ofSEQ ID NO:2); SSTGVLFVKFGPPPTASPG (SEQ ID NO:239; i.e., amino acids141-159 of SEQ ID NO:2); SNPMILMRLKLPNCE (SEQ ID NO:240; i.e., aminoacids 269-283 of SEQ ID NO:2); and the rest of the extracellularsequence of SEQ ID NO:2; or a functional fragment or variant of any ofthese epitopes.

In some embodiments, an anti-ROR1 antibody is provided that binds to apeptide having the sequence KNDAPVVQEPRRLSFRSTIYGSR (SEQ ID NO:237;i.e., amino acids 93-115 of SEQ ID NO:2). In some embodiments, ananti-ROR1 antibody is provided that binds to a peptide having thesequence AANCIRIGIPMADPI (SEQ ID NO:238; i.e., amino acids 293-307 ofSEQ ID NO:2). In some embodiments, an anti-ROR1 antibody is providedthat binds to a peptide having the sequence SSTGVLFVKFGPPPTASPG (SEQ IDNO:239; i.e., amino acids 141-159 of SEQ ID NO:2). In some embodiments,an anti-ROR1 antibody is provided that binds to a peptide having thesequence SNPMILMRLKLPNCE (SEQ ID NO:240; i.e., amino acids 269-283 ofSEQ ID NO:2).

In some embodiments, an anti-ROR1 antibody is provided that competes forbinding to ROR1 with antibody 601-3-2. In some embodiments, an anti-ROR1antibody is provided that competes for binding to ROR1 with antibody601-3-12. In some embodiments, an anti-ROR1 antibody is provided thatcompetes for binding to ROR1 with antibody 601-3-16.

The inventors have also determined the Complementarity DeterminingRegions (CDRs) of both the heavy and light chains of the ROR1-specificantibodies of the invention, and have found highly conserved motifswithin each of these four epitope classes. Surprisingly, as illustratedin FIGS. 8-11, they found that there are three sub-groups in epitopeclass I (i.e., sub-groups Ia, Ib and Ic), five sub-groups in epitopeclass II (i.e., sub-groups IIa, IIb, IIc and IId), and two sub-groups ineach of epitope classes III (i.e., sub-groups IIIa and IIIb) and IV(i.e., sub-groups IVa and IVb) to which embodiments of the antibody canbind. In some embodiments, an antibody is provided that competes forROR1 binding with an antibody in epitope class I (e.g., competes with anantibody in sub-group Ia, Ib or Ic). In some embodiments, an antibody isprovided that competes for ROR1 binding with an antibody in epitopeclass II (e.g., competes with an antibody in sub-group IIa, IIb, IIc orIId. In some embodiments, an antibody is provided that competes for ROR1binding with an antibody in epitope class III (e.g., competes with anantibody in sub-group IIIa or IIIb). In some embodiments, an antibody isprovided that competes for ROR1 binding with an antibody in epitopeclass IV (e.g., competes with an antibody in sub-group IVa or IVb).

As described in Example 2, the inventors have isolated a total of 45antibodies, which are immunospecific for ROR1, 14 of which arepreferred. These 14 preferred antibodies: (i) represent all of theabove-mentioned epitope classes and their sub-groups, (ii) have highROR1-binding affinity; and (iii) form a pool that can be converted intofull IgG molecules. All of the antibodies described herein can bedeveloped for therapeutic and diagnostic use and so are clearlyvaluable.

In some embodiments, and anti-ROR1 antibody is provided, wherein theantibody comprises the heavy chain variable region and the light chainvariable region of an anti-ROR1 antibody selected from Antibody 601-1;Antibody 601-2 (3-12); Antibody 601-3 (3-16); Antibody 601-4; Antibody601-5 (3-2); Antibody 601-6; Antibody 601-9; Antibody 601-13; Antibody601-14; Antibody 601-17; Antibody 601-18Antibody 601-28; Antibody601-37; Antibody 601-40; Antibody 601-43; Antibody 601-50; Antibody601-51; Antibody 601-56; Antibody 601-57; Antibody 601-65; Antibody601-66; Antibody 601-69; Antibody 601-70; Antibody 601-81; Antibody601-86; Antibody 601-87; Antibody 601-100; Antibody 601-101; Antibody601-102; Antibody 601-103; Antibody 601-108; Antibody 601-109; Antibody601-110; Antibody 601-112; Antibody 601-119; Antibody 601-120; Antibody601-128; Antibody 601-130; Antibody 601-134; Antibody 601-136; Antibody601-137; Antibody 601-141; Antibody 601-147; Antibody 601-149; orAntibody 601-153. The heavy chain and light chain variable regions ofthose antibodies are shown, for example, in the tables herein titled“Table of Certain Light Chain Variable Region Sequences” and “Table ofCertain Heavy Chain Variable Region Sequences.”

In some embodiments, and anti-ROR1 antibody is provided, wherein theantibody comprises the heavy chain variable region that is at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to aheavy chain variable region of anti-ROR1 antibody selected from Antibody601-1; Antibody 601-2 (3-12); Antibody 601-3 (3-16); Antibody 601-4;Antibody 601-5 (3-2); Antibody 601-6; Antibody 601-9; Antibody 601-13;Antibody 601-14; Antibody 601-17; Antibody 601-18; Antibody 601-28;Antibody 601-37; Antibody 601-40; Antibody 601-43; Antibody 601-50;Antibody 601-51; Antibody 601-56; Antibody 601-57; Antibody 601-65;Antibody 601-66; Antibody 601-69; Antibody 601-70; Antibody 601-81;Antibody 601-86; Antibody 601-87; Antibody 601-100; Antibody 601-101;Antibody 601-102; Antibody 601-103; Antibody 601-108; Antibody 601-109;Antibody 601-110; Antibody 601-112; Antibody 601-119; Antibody 601-120;Antibody 601-128; Antibody 601-130; Antibody 601-134; Antibody 601-136;Antibody 601-137; Antibody 601-141; Antibody 601-147; Antibody 601-149;or Antibody 601-153. In some such embodiments, the antibody comprisesthe heavy chain CDRs of the reference antibody. The heavy chain CDRs ofeach of those antibodies are shown, for example, in the table hereintitled “Table of Certain Heavy Chain CDR Sequences.”

In some embodiments, and anti-ROR1 antibody is provided, wherein theantibody comprises the light chain variable region that is at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to alight chain variable region of anti-ROR1 antibody selected from Antibody601-1; Antibody 601-2 (3-12); Antibody 601-3 (3-16); Antibody 601-4;Antibody 601-5 (3-2); Antibody 601-6; Antibody 601-9; Antibody 601-13;Antibody 601-14; Antibody 601-17; Antibody 601-18Antibody 601-28;Antibody 601-37; Antibody 601-40; Antibody 601-43; Antibody 601-50;Antibody 601-51; Antibody 601-56; Antibody 601-57; Antibody 601-65;Antibody 601-66; Antibody 601-69; Antibody 601-70; Antibody 601-81;Antibody 601-86; Antibody 601-87; Antibody 601-100; Antibody 601-101;Antibody 601-102; Antibody 601-103; Antibody 601-108; Antibody 601-109;Antibody 601-110; Antibody 601-112; Antibody 601-119; Antibody 601-120;Antibody 601-128; Antibody 601-130; Antibody 601-134; Antibody 601-136;Antibody 601-137; Antibody 601-141; Antibody 601-147; Antibody 601-149;or Antibody 601-153. In some such embodiments, the antibody comprisesthe light chain CDRs of the reference antibody. The light chain CDRs ofeach of those antibodies are shown, for example, in the table hereintitled “Table of Certain Light Chain CDR Sequences.”

FIGS. 8-11 show that the inventors have determined consensus sequencesfor the variable regions of the heavy and light chains of the isolatedantibodies, which form the CDRs (L1, L2 and L3 being the three CDRs ofthe light chain, and H1, H2 and H3 being the three CDRs of the heavychain). Clearly, these conserved residues are important for defining theantibodies' binding specificity and affinity for the ROR1 protein,preferably the extracellular domain (ECD) thereof, and hence provideuseful information for antibody engineering in order to modify andimprove the binding profile of the antibody. The inventors havedetermined the amino acid and DNA sequences of each of the preferred 14antibodies, as described in Example 2.

Thus, in one embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region comprising an amino acidsequence selected from a group consisting of: (i) SEQ ID NO:8; (ii) SEQID NO:9; (iii) SEQ ID NO:10; (iv) SEQ ID NO:16; (v) SEQ ID NO:17; and(vi) SEQ ID NO:18.

In another embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region comprising an amino acidsequence selected from a group consisting of: (i) SEQ ID NO:24; (ii) SEQID NO:25; (iii) SEQ ID NO:26; (iv) SEQ ID NO:32; (v) SEQ ID NO:33; and(vi) SEQ ID NO:34.

In another embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region comprising an amino acidsequence selected from a group consisting of: (i) SEQ ID NO:40; (ii) SEQID NO:41; (iii) SEQ ID NO:42; (iv) SEQ ID NO:48; (v) SEQ ID NO:49; and(vi) SEQ ID NO:50.

In another embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region comprising an amino acidsequence selected from a group consisting of: (i) SEQ ID NO:56; (ii) SEQID NO:57; (iii) SEQ ID NO:58; (iv) SEQ ID NO:64; (v) SEQ ID NO:65; and(vi) SEQ ID NO:66.

In another embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region comprising an amino acidsequence selected from a group consisting of: (i) SEQ ID NO:72; (ii) SEQID NO:73; (iii) SEQ ID NO:74; (iv) SEQ ID NO:80; (v) SEQ ID NO:81; and(vi) SEQ ID NO:82.

In another embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region comprising an amino acidsequence selected from a group consisting of: (i) SEQ ID NO:88; (ii) SEQID NO:89; (iii) SEQ ID NO:90; (iv) SEQ ID NO:96; (v) SEQ ID NO:97; and(vi) SEQ ID NO:98.

In another embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region comprising an amino acidsequence selected from a group consisting of: (i) SEQ ID NO:104; (ii)SEQ ID NO:105; (iii) SEQ ID NO:106; (iv) SEQ ID NO:112; (v) SEQ IDNO:113; and (vi) SEQ ID NO:114.

In another embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region comprising an amino acidsequence selected from a group consisting of: (i) SEQ ID NO:120; (ii)SEQ ID NO:121; (iii) SEQ ID NO:122;(iv) SEQ ID NO:128; (v) SEQ IDNO:129; and (vi) SEQ ID NO:130.

In another embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region comprising an amino acidsequence selected from a group consisting of: (i) SEQ ID NO:136; (ii)SEQ ID NO:137; (iii) SEQ ID NO:138; (iv) SEQ ID NO:144; (v) SEQ IDNO:145; and (vi) SEQ ID NO:146.

In another embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region comprising an amino acidsequence selected from a group consisting of: (i) SEQ ID NO:152; (ii)SEQ ID NO:153; (iii) SEQ ID NO:154; (iv) SEQ ID NO:160; (v) SEQ IDNO:161; and (vi) SEQ ID NO:162.

In another embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region comprising an amino acidsequence selected from a group consisting of: (i) SEQ ID NO:168; (ii)SEQ ID NO:169; (iii) SEQ ID NO:170; (iv) SEQ ID NO:176; (v) SEQ IDNO:177; and (vi) SEQ ID NO:178.

In another embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region comprising an amino acidsequence selected from a group consisting of: (i) SEQ ID NO:184; (ii)SEQ ID NO:185; (iii) SEQ ID NO:186;

(iv) SEQ ID NO:192; (v) SEQ ID NO:193; and (vi) SEQ ID NO:194.

In another embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region comprising an amino acidsequence selected from a group consisting of: (i) SEQ ID NO:200; (ii)SEQ ID NO:201; (iii) SEQ ID NO:202; (iv) SEQ ID NO:208; (v) SEQ IDNO:209; and (vi) SEQ ID NO:210.

In another embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region comprising an amino acidsequence selected from a group consisting of: (i) SEQ ID NO:216; (ii)SEQ ID NO:217; (iii) SEQ ID NO:218; (iv) SEQ ID NO:224; (v) SEQ IDNO:225; and (vi) SEQ ID NO:226.

In one embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region encoded by a nucleic acidcomprising a nucleotide sequence selected from a group consisting of:(i) SEQ ID NO:5; (ii) SEQ ID NO:6; (iii) SEQ ID NO:7; (iv) SEQ ID NO:13;(v) SEQ ID NO:14; and (vi) SEQ ID NO:15.

In another embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region encoded by a nucleic acidcomprising a nucleotide sequence selected from a group consisting of:(i) SEQ ID NO:21; (ii) SEQ ID NO:22; (iii) SEQ ID NO:23; (iv) SEQ IDNO:29; (v) SEQ ID NO:30; and (vi) SEQ ID NO:31.

In one embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region encoded by a nucleic acidcomprising a nucleotide sequence selected from a group consisting of:(i) SEQ ID NO:37; (ii) SEQ ID NO:38; (iii) SEQ ID NO:39; (iv) SEQ IDNO:45; (v) SEQ ID NO:46; and (vi) SEQ ID NO:47.

In one embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region encoded by a nucleic acidcomprising a nucleotide sequence selected from a group consisting of:(i) SEQ ID NO:53; (ii) SEQ ID NO:54; (iii) SEQ ID NO:55; (iv) SEQ IDNO:61; (v) SEQ ID NO:62; and (vi) SEQ ID NO:63.

In one embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region encoded by a nucleic acidcomprising a nucleotide sequence selected from a group consisting of:(i) SEQ ID NO:69; (ii) SEQ ID NO:70; (iii) SEQ ID NO:71; (iv) SEQ IDNO:77; (v) SEQ ID NO:78; and (vi) SEQ ID NO:79.

In one embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region encoded by a nucleic acidcomprising a nucleotide sequence selected from a group consisting of:(i) SEQ ID NO:85; (ii) SEQ ID NO:86; (iii) SEQ ID NO:87; (iv) SEQ IDNO:93; (v) SEQ ID NO:94; and (vi) SEQ ID NO:95.

In one embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region encoded by a nucleic acidcomprising a nucleotide sequence selected from a group consisting of:(i) SEQ ID NO:101; (ii) SEQ ID NO:102; (iii) SEQ ID NO:103; (iv) SEQ IDNO:109; (v) SEQ ID NO:110; and (vi) SEQ ID NO:111.

In one embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region encoded by a nucleic acidcomprising a nucleotide sequence selected from a group consisting of:(i) SEQ ID NO:117; (ii) SEQ ID NO:118; (iii) SEQ ID NO:119; (iv) SEQ IDNO:125; (v) SEQ ID NO:126; and (vi) SEQ ID NO:127.

In one embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region encoded by a nucleic acidcomprising a nucleotide sequence selected from a group consisting of:(i) SEQ ID NO:133; (ii) SEQ ID NO:134; (iii) SEQ ID NO:135; (iv) SEQ IDNO:141; (v) SEQ ID NO:142; and (vi) SEQ ID NO:143.

In one embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region encoded by a nucleic acidcomprising a nucleotide sequence selected from a group consisting of:(i) SEQ ID NO:149; (ii) SEQ ID NO:150; (iii) SEQ ID NO:151; (iv) SEQ IDNO:157; (v) SEQ ID NO:158; and (vi) SEQ ID NO:159.

In one embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region encoded by a nucleic acidcomprising a nucleotide sequence selected from a group consisting of:(i) SEQ ID NO:165; (ii) SEQ ID NO:166; (iii) SEQ ID NO:167; (iv) SEQ IDNO:173; (v) SEQ ID NO:174; and (vi) SEQ ID NO:175.

In one embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region encoded by a nucleic acidcomprising a nucleotide sequence selected from a group consisting of:(i) SEQ ID NO:181; (ii) SEQ ID NO:182; (iii) SEQ ID NO:183; (iv) SEQ IDNO:189; (v) SEQ ID NO:190; and (vi) SEQ ID NO:191.

In one embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region encoded by a nucleic acidcomprising a nucleotide sequence selected from a group consisting of:(i) SEQ ID NO:197; (ii) SEQ ID NO:198; (iii) SEQ ID NO:199; (iv) SEQ IDNO:205; (v) SEQ ID NO:206; and (vi) SEQ ID NO:207.

In one embodiment, the antibody or functional fragment thereof maycomprise at least one antigen binding region encoded by a nucleic acidcomprising a nucleotide sequence selected from a group consisting of:(i) SEQ ID NO:213; (ii) SEQ ID NO:214; (iii) SEQ ID NO:215; (iv) SEQ IDNO:221; (v) SEQ ID NO:222; and (vi) SEQ ID NO:223.

It will be appreciated that the antigen binding region may comprises aComplementarily Determining Region (CDR) of the antibody, or functionalfragment thereof, and that mutations may reside in framework regionsbetween the CDRs of the antibody or functional fragment thereof. Therecombinant immunoglobulin may comprise any, or all, of the antigenbinding regions described herein. The recombinant immunoglobulin maycomprise an antigen binding site with which the antigen binds,preferably eliciting an immunological response. Preferably, the at leastone antigen binding region forms at least part of the antigen bindingsite. The antibody or functional fragment thereof may comprise at leasttwo, suitably at least three, more suitably at least four antigenbinding regions defined in the first aspect. The antibody or functionalfragment may comprise at least five, more preferably at least sixantigen binding regions. The antibody or fragment thereof may thereforecomprise at least one, two, three, four, five or six amino acidsequences defined in (i) to (vi) in any embodiment of antibody describedherein. Preferably, the antibody or fragment thereof comprises all of(i) to (vi) in any embodiment.

In one embodiment of the antibody or a functional fragment thereof, thepolypeptide sequence of the variable region of the light chain maycomprise an amino acid sequence substantially as set out in SEQ ID NO:4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196 or 212, or afunctional variant or fragment thereof.

The polypeptide sequence of the variable region of the heavy chain ofthe antibody or functional fragment thereof may comprise an amino acidsequence substantially as set out in SEQ ID NO: 12, 28, 44, 60, 76, 92,108, 124, 140, 156, 172, 188, 204 or 220, or a functional variant orfragment thereof.

The antibody or functional fragment thereof may comprise a light chainvariable region (VL) and/or a heavy chain variable region (VH), thelight chain variable region comprising an amino acid sequence which issubstantially as set out in SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116,132, 148, 164, 180, 196 or 212, or a functional fragment or variantthereof, the heavy chain variable region comprising the amino acidsequence which is substantially as set out in SEQ ID NO: 12, 28, 44, 60,76, 92, 108, 124, 140, 156, 172, 188, 204 or 220, or a functionalfragment or variant thereof.

In one embodiment of the antibody or a functional fragment thereof, theDNA sequence of the variable region of the light chain may comprise anucleotide sequence substantially as set out in SEQ ID NO: 3, 19, 35,51, 67, 83, 99, 115, 131, 147, 163, 179, 195 or 211, or a functionalvariant or fragment thereof. In one embodiment of the antibody or afunctional fragment thereof, the DNA sequence of the variable region ofthe heavy chain may comprise a nucleotide sequence substantially as setout in SEQ ID NO: 11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187,203 or 219, or a functional variant or fragment thereof.

The antibody or functional fragment thereof may comprise a light chainvariable region (VL) and/or a heavy chain variable region (VH), thelight chain variable region being encoded by a polynucleotide comprisinga nucleotide sequence which is substantially as set out in SEQ ID NO: 3,19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195 or 211, or afunctional fragment or variant thereof, the heavy chain variable regionbeing encoded by a polynucleotide comprising a nucleotide sequence whichis substantially as set out in SEQ ID NO: 11, 27, 43, 59, 75, 91, 107,123, 139, 155, 171, 187, 203 or 219, or a functional fragment or variantthereof.

The polypeptide sequence of the constant region of the light chain ofone embodiment of the antibody of the invention may comprise an aminoacid sequence which is substantially as set out in SEQ ID NO: 227, or afunctional fragment or variant thereof. The polypeptide sequence of theconstant region of the heavy chain of one embodiment of the antibody ofthe invention may comprise an amino acid sequence which is substantiallyas set out in SEQ ID NO: 228, or a functional fragment or variantthereof.

The DNA sequence encoding the constant region of the light chain of oneembodiment of the antibody of the invention may comprise a nucleotidesequence which is substantially as set out in SEQ ID NO: 229, or afunctional fragment or variant thereof. The DNA sequence encoding theconstant region of the heavy chain of one embodiment of the antibody ofthe invention may comprise a nucleotide sequence which is substantiallyas set out in SEQ ID NO: 230, or a functional fragment or variantthereof.

According to a third aspect, there is provided an isolated peptidecapable of binding to Receptor Tyrosine Kinase-Like Orphan Receptor 1(ROR1) protein, the peptide comprising an amino acid sequence selectedfrom a group consisting of:

-   -   (i) SEQ ID NO:8, 9, 10, 16, 17 and/or 18;    -   (ii) SEQ ID NO:24, 25, 26, 32, 33 and/or 34;    -   (iii) SEQ ID NO:40, 41, 42, 48, 49 and/or 50;    -   (iv) SEQ ID NO:56, 57, 58, 64, 65 and/or 66;    -   (v) SEQ ID NO:72, 73, 74, 80, 81 and/or 82;    -   (vi) SEQ ID NO:88, 89, 90, 96, 97 and/or 98;    -   (vii) SEQ ID NO:104, 105, 106, 112, 113 and/or 114;    -   (viii) SEQ ID NO:120, 121, 122, 128, 129 and/or 130;    -   (ix) SEQ ID NO:136, 137, 138, 144, 145 and/or 146;    -   (x) SEQ ID NO:152, 153, 154, 160, 161 and/or 162;    -   (xi) SEQ ID NO:168, 169, 170, 176, 177 and/or 178;    -   (xii) SEQ ID NO:184, 185, 186, 192, 193 and/or 194;    -   (xiii) SEQ ID NO:200, 201, 202, 208, 209 and/or 210; and/or    -   (xiv) SEQ ID NO:216, 217, 218, 224, 225 and/or 226.

The peptide of the third aspect is preferably capable of binding to anextracellular domain of ROR1 protein. Thus, the isolated peptide of thethird aspect may be an anti-Receptor Tyrosine Kinase-Like OrphanReceptor 1 (ROR1) antibody, or a functional fragment thereof. Theantibody may or may not be human. For example, the antibody or fragmentthereof may be murine. It may also be recombinant. It will beappreciated that the amino acid sequences defined in each of (i) to(xiv) of the third aspect are the CDRs of the antibody or a functionalfragment thereof of the first aspect. The isolated peptide may thereforecomprise at least two, three, four, five or six amino acid sequencesdefined in any of (i) to (xiv). Preferably, the peptide comprises all ofthe amino acid sequences defined in any of (i) to (xiv).

The peptide may comprise an amino acid sequence substantially as set outin SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196or 212, or a functional variant or fragment thereof. The peptide maycomprise an amino acid sequence substantially as set out in SEQ ID NO:12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204 or 220, or afunctional variant or fragment thereof. The peptide may comprise anamino acid sequence which is substantially as set out in SEQ ID NO: 227or 228, or a functional fragment or variant thereof.

According to a fourth aspect, there is provided an isolated nucleic acidencoding a peptide capable of binding to Receptor Tyrosine Kinase-LikeOrphan Receptor 1 (ROR1) protein, the nucleic acid comprising anucleotide sequence selected from a group consisting of:

-   -   (i) SEQ ID NO:5, 6, 7, 13, 14 and/or 15;    -   (ii) SEQ ID NO:21, 22, 23, 29, 30 and/or 31;    -   (iii) SEQ ID NO:37, 38, 39, 45, 46 and/or 47;    -   (iv) SEQ ID NO:53, 54, 55, 61, 62 and/or 63;    -   (v) SEQ ID NO:69, 70, 71, 77, 78 and/or 79;    -   (vi) SEQ ID NO:85, 86, 87, 93, 94 and/or 95;    -   (vii) SEQ ID NO:101, 102, 103, 109, 110 and/or 111;    -   (viii) SEQ ID NO:117, 118, 119, 125, 126 and/or 127;    -   (ix) SEQ ID NO:133, 134, 135, 141, 142 and/or 143;    -   (x) SEQ ID NO:149, 150, 151, 157, 158 and/or 159;    -   (xi) SEQ ID NO:165, 166, 167, 173, 174 and/or 175;    -   (xii) SEQ ID NO:181, 182, 183, 189, 190 and/or 191;    -   (xiii) SEQ ID NO:197, 198, 199, 205, 206 and/or 207; and/or    -   (xiv) SEQ ID NO:213, 214, 215, 221, 222 and/or 223.

Preferably, the nucleic acid may encode an anti-Receptor TyrosineKinase-Like Orphan Receptor 1 (ROR1) antibody, or a functional fragmentthereof. The antibody may or may not be human. For example, the antibodymay be murine. It may also be recombinant. It will be appreciated thatthe nucleotide sequences defined in each of (i) to (xiv) of the fourthaspect encode the CDRs of the antibody or a functional fragment thereofof the first aspect. Thus, the nucleic acid may comprise at least two,three, four, five or six nucleotide sequences defined in any of (i) to(xiv). Preferably, the nucleic acid comprises all of the nucleotidesequences defined in any of (i) to (vi). Preferably, the nucleic acidcomprises a nucleotide sequence substantially encoding an amino acidsequence of at least one antigen binding region of the human antibody,or functional fragment thereof.

The nucleic acid may comprise a nucleotide sequence substantially as setout in SEQ ID NO: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179,195 or 211, or a functional variant or fragment thereof. The nucleicacid may comprise a nucleotide sequence substantially as set out in SEQID NO: 11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203 or 219,or a functional variant or fragment thereof. The nucleic acid maycomprise a nucleotide sequence which is substantially as set out in SEQID NO: 229 or 230, or a functional fragment or variant thereof.

Advantageously, the antibody or functional fragment thereof has utilityof a therapeutic agent in its own right, and is a significantimprovement on therapies which use antibodies comprising a non-humanregion (e.g., murine), such as a Fc fragment or framework regions, or atleast one murine antigen binding region or Complementarity DeterminingRegion (CDR). However, in addition, technologies to maximize drugefficacy have been evaluated, including glycosylation engineering toenhance the ADCC (Antibody-Dependent Cell-Mediated Cytotoxicity) and/orCDC (Complement-Dependent Cytotoxicity) activity of the antibody orfunctional fragment thereof, conjugation to a cytotoxic moiety, such asradiation, a cytotoxic drug or toxin, and generation of a bispecificantibody with one arm targeting a tumor cell, and the other armattracting cytotoxic T cells.

Thus, in a fifth aspect, there is provided an antibody-drug conjugate(ADC) comprising the antibody or a functional fragment thereof of thefirst aspect, and a cytotoxic moiety.

Antibody-drug conjugates (ADCs) can be used to deliver a potentcytotoxic drug selectively to a target cell via an antibody. Suchmethods, when applied to a tumor antigen target, can enhance theantitumor activity of antibodies and improve the tumor-to-normal tissueselectivity of chemotherapy. One key parameter for ADC development isthat the antibody can be endocytosed once bound to target antigen, andtherefore, deliver the conjugated drug into target cancer cells. FIG. 26shows that the antibody of the invention is effectively endocytosed.

The cytotoxic moiety may be a toxin, such as monomethyl auristatin E(MMAE), monomethyl auristatin F (MMAF) or maytansine. The drug moietymay be an alpha-emitting radionucleotide, such as a 225Ac label. Thesetoxins can be linked to the antibody or functional fragment thereof(i.e., an antigen-binding fragment thereof) via a cleavable linker, suchas a disulfide bond, a hydrazone linker or a peptide linker, or via anon-cleavable linkers, such as a thioether bond using a SMCC(N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate) linker.

According to a sixth aspect, there is provided an antibody or afunctional fragment thereof as defined in the first aspect, a peptide asdefined in the third aspect, a nucleic acid as defined in the fourthaspect, or an antibody-drug conjugate as defined in the fifth aspect,each being optionally derivatized, for use in therapy or in diagnosis.

The antibody or functional fragment thereof as defined in the firstaspect, the peptide as defined in the third aspect, the nucleic acid asdefined in the fourth aspect, or the antibody-drug conjugate as definedin the fifth aspect, may be used as a medicament, which is preferablyadapted for use in the treatment, amelioration or prevention of cancer.

Therefore, according to a seventh aspect, there is provided an antibodyor a functional fragment thereof as defined in the first aspect, apeptide as defined in the third aspect, a nucleic acid as defined in thefourth aspect, or an antibody-drug conjugate as defined in the fifthaspect, each being optionally derivatized, for use in treating,preventing or ameliorating cancer.

The term “derivatized” can mean that the antibody or functional fragmentthereof, peptide, nucleic acid or conjugate may be modified prior touse, preferably to produce a derivative or variant thereof. Examples ofderivatization may include PEGylated antibodies or PEGylated antibodyfragments, or antibody-cytokine fusion proteins. However, in someembodiments, the antibody or functional fragment thereof, peptide,nucleic acid or conjugate may not be derivatized.

ROR1 is expressed in a wide range of human cancer types, and so theantibody or functional fragment thereof, peptide, a nucleic acid orconjugate may be used in the treatment, prevention, amelioration ordiagnosis of a ROR1-positive cancer type.

Non-limiting exemplary cancers that may be treated with an anti-ROR1antibody include carcinoma, lymphoma, blastoma, sarcoma, and leukemia.More particular non-limiting examples of such cancers include squamouscell cancer, small-cell lung cancer, pituitary cancer, esophagealcancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, renal cancer, liver cancer, prostate cancer,vulval cancer, thyroid cancer, hepatic carcinoma, brain cancer,neuroblastoma, endometrial cancer, testis cancer, cholangiocarcinoma,gallbladder carcinoma, gastric cancer, melanoma, and various types ofhead and neck cancer. In some embodiments, lung cancer is non-small celllung cancer or lung squamous cell carcinoma. In some embodiments,leukemia is acute myeloid leukemia, B-cell acute lymphoblastic leukemia(B-ALL); or chronic lymphocytic leukemia (CLL). In some embodiments,lymphoma is mantle cell lymphoma (MCL) or marginal zone lymphoma (MZL).In some embodiments, breast cancer is breast invasive carcinoma. In someembodiments, ovarian cancer is ovarian serous cystadenocarcinoma. Insome embodiments, kidney cancer is kidney renal clear cell carcinoma. Insome embodiments, colon cancer is colon adenocarcinoma. In someembodiments, bladder cancer is bladder urothelial carcinoma. In any ofthe cancers described above, the cancer may be an ROR1-positive cancer.

According to an eighth aspect, there is provided a method of treating,preventing or ameliorating cancer in a subject, the method comprisingadministering, to a patient in need of such treatment, a therapeuticallyeffective amount of an antibody or a functional fragment thereof asdefined in the first aspect, a peptide as defined in the third aspect, anucleic acid as defined in the fourth aspect, or an antibody-drugconjugate as defined in the fifth aspect, each being optionallyderivatized.

It will be appreciated that antibodies, fragments, peptides, nucleicacids and conjugates according to the invention (collectively referredto herein as “agents”) may be used in a monotherapy (e.g., the use of anantibody or fragment thereof alone, or the use of the antibody-drugconjugate alone), for treating, ameliorating or preventing cancer.Alternatively, agents according to the invention may be used as anadjunct to, or in combination with, known therapies for treating,ameliorating, or preventing cancer. Such therapies include, but are notlimited to, chemotherapy, anti-angiogenesis agents, growth inhibitoryagents, immunotherapy, radiation therapy, etc.

Nonlimiting exemplary chemotherapeutic agents that may be combined withthe anti-ROR1 antibodies described herein include, but are not limitedto, alkylating agents such as thiotepa and Cytoxan® cyclosphosphamide;alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammal I and calicheamicinomegall (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., Taxol®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), Abraxane®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTaxotere® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;Gemzar® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; Navelbine® vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluorometlhylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g.,erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Further nonlimiting exemplary chemotherapeutic agents includeanti-hormonal agents that act to regulate or inhibit hormone action oncancers such as anti-estrogens and selective estrogen receptormodulators (SERMs), including, for example, tamoxifen (includingNolvadex® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and Fareston® toremifene;aromatase inhibitors that inhibit the enzyme aromatase, which regulatesestrogen production in the adrenal glands, such as, for example,4(5)-imidazoles, aminoglutethimide, Megase® megestrol acetate, Aromasin®exemestane, formestanie, fadrozole, Rivisor® vorozole, Femara®letrozole, and Arimidex® anastrozole; and anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as wellas troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those which inhibit expression of genesin signaling pathways implicated in abherant cell proliferation, suchas, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as a VEGFexpression inhibitor (e.g., Angiozyme® ribozyme) and a HER2 expressioninhibitor; vaccines such as gene therapy vaccines, for example,Allovectin® vaccine, Leuvectin® vaccine, and Vaxid® vaccine; Proleukin®rIL-2; Lurtotecan® topoisomerase 1 inhibitor; Abarelix® rmRH; andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Non-limiting exemplary an anti-angiogenesis agents include antibodies orother antagonists to an angiogenic agent, e.g., antibodies to VEGF-A(e.g., bevacizumab (Avastin)) or to the VEGF-A receptor (e.g., KDRreceptor or Flt-1 receptor), anti-PDGFR inhibitors such as Gleevec®(Imatinib Mesylate), small molecules that block VEGF receptor signaling(e.g., PTK787/ZK2284, SU6668, Sutent®/SU11248 (sunitinib malate),AMG706, or those described in, e.g., international patent application WO2004/113304). Anti-angiogensis agents also include native angiogenesisinhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun andD'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003)Oncogene 22:3172-3179 (e.g., Table 3 listing anti-angiogenic therapy inmalignant melanoma); Ferrara & Alitalo (1999) Nature Medicine5(12):1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 (e.g., Table2 listing known anti-angiogenic factors); and, Sato (2003) Int. J. Clin.Oncol. 8:200-206 (e.g., Table 1 listing anti-angiogenic agents used inclinical trials).

Examples of growth inhibitory agents include, but are not limited to,agents that block cell cycle progression (at a place other than Sphase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in Mendelsohn and Israel, eds., TheMolecular Basis of Cancer, Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (W.B. Saunders,Philadelphia, 1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel)are anticancer drugs both derived from the yew tree. Docetaxel(Taxotere®, Rhone-Poulenc Rorer), derived from the European yew, is asemisynthetic analogue of paclitaxel (Taxol®, Bristol-Myers Squibb).Paclitaxel and docetaxel promote the assembly of microtubules fromtubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

Exemplary therapeutic agents that may be combined with the anti-ROR1antibodies described herein include chemotherapeutic agents, growthinhibitory agents, cytotoxic agents, agents used in radiation therapy,anti-angiogenesis agents, cancer immunotherapeutic agents, apoptoticagents, anti-tubulin agents, and other-agents to treat cancer, such asanti-HER-2 antibodies, anti-CD20 antibodies, an epidermal growth factorreceptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor),HER1/EGFR inhibitor (e.g., erlotinib (Tarceva®), platelet derived growthfactor inhibitors (e.g., Gleevec® (Imatinib Mesylate)), a COX-2inhibitor (e.g., celecoxib), interferons, CTLA4 inhibitors (e.g.,anti-CTLA antibody ipilimumab (YERVOY®)), PD-1 inhibitors (e.g.,anti-PD1 antibodies, BMS-936558), PDL1 inhibitors (e.g., anti-PDL1antibodies, MPDL3280A), PDL2 inhibitors (e.g., anti-PDL2 antibodies),TIM3 inhibitors (e.g., anti-TIM3 antibodies), cytokines, antagonists(e.g., neutralizing antibodies) that bind to one or more of thefollowing targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA,PD-1, PDL1, PDL2, CTLA4, TIM3, or VEGF receptor(s), TRAIL/Apo2, andother bioactive and organic chemical agents, etc. Combinations thereofare also included in the invention.

The agents according to the invention may be combined in compositionshaving a number of different forms depending, in particular, on themanner in which the composition is to be used. Thus, for example, thecomposition may be in the form of a powder, tablet, capsule, liquid,ointment, cream, gel, hydrogel, aerosol, spray, micellar solution,transdermal patch, liposome suspension or any other suitable form thatmay be administered to a person or animal in need of treatment. It willbe appreciated that the vehicle of medicaments according to theinvention should be one which is well-tolerated by the subject to whomit is given, and preferably enables delivery of the agents across theblood-brain barrier.

Medicaments comprising agents of the invention may be used in a numberof ways. For instance, oral administration may be required, in whichcase the agents may be contained within a composition that may, forexample, be ingested orally in the form of a tablet, capsule or liquid.Compositions comprising agents and medicaments of the invention may beadministered by inhalation (e.g., intranasally). Compositions may alsobe formulated for topical use. For instance, creams or ointments may beapplied to the skin. Agents and medicaments according to the inventionmay also be incorporated within a slow- or delayed-release device. Suchdevices may, for example, be inserted on or under the skin, and themedicament may be released over weeks or even months. The device may belocated at least adjacent the treatment site. Such devices may beparticularly advantageous when long-term treatment with agents usedaccording to the invention is required and which would normally requirefrequent administration (e.g., at least daily injection).

In a preferred embodiment, agents and medicaments according to theinvention may be administered to a subject by injection into the bloodstream or directly into a site requiring treatment. Injections may beintravenous (bolus or infusion) or subcutaneous (bolus or infusion), orintradermal (bolus or infusion). It will be appreciated that the amountof the antibodies, fragments, peptides and nucleic acids (i.e., agent)that is required is determined by its biological activity andbioavailability, which in turn depends on the mode of administration,the physiochemical properties of the agent, and whether it is being usedas a monotherapy or in a combined therapy. The frequency ofadministration will also be influenced by the half-life of the agentwithin the subject being treated. Optimal dosages to be administered maybe determined by those skilled in the art, and will vary with theparticular agent in use, the strength of the pharmaceutical composition,the mode of administration, and the advancement of the bacterialinfection. Additional factors depending on the particular subject beingtreated will result in a need to adjust dosages, including subject age,weight, gender, diet, and time of administration.

Generally, a daily dose of between 0.001 μg/kg of body weight and 10mg/kg of body weight of agent according to the invention may be used fortreating, ameliorating, or preventing cancer, depending upon whichagent. More preferably, the daily dose of agent is between 0.01 μg/kg ofbody weight and 1 mg/kg of body weight, more preferably between 0.1μg/kg and 100 μg/kg body weight, and most preferably betweenapproximately 0.1 μg/kg and 10 μg/kg body weight.

The agent may be administered before, during or after onset of cancer.Daily doses may be given as a single administration (e.g., a singledaily injection). Alternatively, the agent may require administrationtwice or more times during a day. As an example, agents may beadministered as two (or more depending upon the severity of the cancerinfection being treated) daily doses of between 0.07 g and 700 mg (i.e.,assuming a body weight of 70 kg). A patient receiving treatment may takea first dose upon waking and then a second dose in the evening (if on atwo dose regime) or at 3- or 4-hourly intervals thereafter.Alternatively, a slow release device may be used to provide optimaldoses of agents according to the invention to a patient without the needto administer repeated doses. Known procedures, such as thoseconventionally employed by the pharmaceutical industry (e.g., in vivoexperimentation, clinical trials, etc.), may be used to form specificformulations of the agents according to the invention and precisetherapeutic regimes (such as daily doses of the agents and the frequencyof administration).

In a ninth aspect of the invention, there is provided a pharmaceuticalcomposition comprising an antibody or a functional fragment thereof asdefined in the first aspect, a peptide as defined in the third aspect, anucleic acid as defined in the fourth aspect, or an antibody-drugconjugate as defined in the fifth aspect, each being optionallyderivatized; and optionally a pharmaceutically acceptable vehicle.

The composition may be an anti-cancer composition.

The antibody or a functional fragment thereof, peptide or a nucleic acidmay not be derivatized.

The invention also provides in a tenth aspect, a process for making thecomposition according to the ninth aspect, the process comprisingcombining a therapeutically effective amount of an antibody or afunctional fragment thereof as defined in the first aspect, a peptide asdefined in the third aspect, a nucleic acid as defined in the fourthaspect, or an antibody-drug conjugate as defined in the fifth aspect,each being optionally derivatized, with a pharmaceutically acceptablevehicle.

The antibody or fragment thereof may be as defined with respect to thefirst aspect.

In some embodiments, a therapeutically effective amount of antibody orfragment thereof used may be from about 0.001 ng to about 1 mg, andpreferably from about 0.01 ng to about 100 ng. It is preferred that theamount of antibody or fragment is an amount from about 0.1 ng to about10 ng, and most preferably from about 0.5 ng to about 5 ng.

In one embodiment, the pharmaceutically acceptable vehicle may be asolid, and the composition may be in the form of a powder or tablet. Asolid pharmaceutically acceptable vehicle may include one or moresubstances which may also act as flavoring agents, lubricants,solubilizers, suspending agents, dyes, fillers, glidants, compressionaids, inert binders, sweeteners, preservatives, dyes, coatings, ortablet-disintegrating agents. The vehicle may also be an encapsulatingmaterial. In powders, the vehicle is a finely divided solid that is inadmixture with the finely divided active agents according to theinvention. In tablets, the active agent may be mixed with a vehiclehaving the necessary compression properties in suitable proportions andcompacted in the shape and size desired. The powders and tabletspreferably contain up to 99% of the active agents. Suitable solidvehicles include, for example calcium phosphate, magnesium stearate,talc, sugars, lactose, dextrin, starch, gelatin, cellulose,polyvinylpyrrolidine, low melting waxes and ion exchange resins. Inanother embodiment, the pharmaceutical vehicle may be a gel and thecomposition may be in the form of a cream or the like.

However, the pharmaceutical vehicle may be a liquid, and thepharmaceutical composition is in the form of a solution. Liquid vehiclesare used in preparing solutions, suspensions, emulsions, syrups, elixirsand pressurized compositions. The active agent according to theinvention may be dissolved or suspended in a pharmaceutically acceptableliquid vehicle such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid vehicle can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, colors, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid vehicles fororal and parenteral administration include water (partially containingadditives as above, e.g., cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g., glycols) and their derivatives,and oils (e.g., fractionated coconut oil and arachis oil). Forparenteral administration, the vehicle can also be an oily ester such asethyl oleate and isopropyl myristate. Sterile liquid vehicles are usefulin sterile liquid form compositions for parenteral administration. Theliquid vehicle for pressurized compositions can be a halogenatedhydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions, which are sterile solutions orsuspensions, can be utilized by, for example, intramuscular,intrathecal, epidural, intraperitoneal, intravenous and particularlysubcutaneous injection. The agent may be prepared as a sterile solidcomposition that may be dissolved or suspended at the time ofadministration using sterile water, saline, or other appropriate sterileinjectable medium.

The agents and compositions of the invention may be administered orallyin the form of a sterile solution or suspension containing other solutesor suspending agents (for example, enough saline or glucose to make thesolution isotonic), bile salts, acacia, gelatin, sorbitan monoleate,polysorbate 80 (oleate esters of sorbitol and its anhydridescopolymerized with ethylene oxide) and the like. The agents usedaccording to the invention can also be administered orally either inliquid or solid composition form. Compositions suitable for oraladministration include solid forms, such as pills, capsules, granules,tablets, and powders, and liquid forms, such as solutions, syrups,elixirs, and suspensions. Forms useful for parenteral administrationinclude sterile solutions, emulsions, and suspensions.

The invention also provides a kit for diagnosing patients suffering fromcancer. Hence, according to an eleventh aspect of the invention, thereis provided a kit for diagnosing a subject suffering from cancer, or apre-disposition thereto, or for providing a prognosis of the subject'scondition, the kit comprising detection means for detecting theconcentration of antigen present in a sample from a test subject,wherein the detection means comprising an antibody or functionalfragment thereof as defined by the first aspect, a peptide as defined bythe third aspect, or a nucleic acid as defined by the fourth aspect,each being optionally derivatized, wherein presence of antigen in thesample suggests that the subject suffers from cancer.

According to a twelfth aspect, there is provided a method for diagnosinga subject suffering from cancer, or a pre-disposition thereto, or forproviding a prognosis of the subject's condition, the method comprisingdetecting the concentration of antigen present in a sample obtained froma subject, wherein the detection is achieved using an antibody orfunctional fragment thereof as defined by the first aspect, a peptide asdefined by the third aspect, or a nucleic acid as defined by the fourthaspect, each being optionally derivatized, and wherein presence ofantigen in the sample suggests that the subject suffers from cancer.

In some embodiments, methods of selecting a patient for treatment withthe anti-ROR1 antibodies described herein comprise determining whetherthe subject has an ROR1-expressing cancer. Such methods may comprisedetecting the presence of ROR1 on cancer cells from the patient, forexample, using an anti-ROR1 antibody described herein.

Preferably, the antigen comprises ROR1 protein, more preferably anextracellular domain thereof. The sample may comprise blood, urine,tissue etc.

Preferably, the kit or method is used to identify the presence orabsence of ROR1-positive cells in the sample, or determine theconcentration thereof in the sample. The term “ROR1-positive cells” canmean a cell expressing ROR1 on its surface. The detection means maycomprise an assay adapted to detect the presence and/or absence ofROR1-positive cells in the sample. The kit or method may comprise theuse of a positive control and/or a negative control against which theassay may be compared. For example, the kit may comprise a reference forthe concentration of ROR1-positive cells in a sample from an individualwho does (i.e., positive control) or does not (i.e., a negative control)suffer from cancer. The kit may further comprise a label which may bedetected. The term “label” can mean a moiety that can be attached to theantibody, fragment, peptide or nucleic acid. Moieties can be used, forexample, for therapeutic or diagnostic procedures. Therapeutic labelsinclude, for example, moieties that can be attached to an antibody orfragment thereof of the invention and used to monitor the binding of theantibody to an ROR1 protein. Diagnostic labels include, for example,moieties which can be detected by analytical methods. Analytical methodsinclude, for example, qualitative and quantitative procedures.Qualitative analytical methods include, for example,immunohistochemistry and indirect immunofluorescence. Quantitativeanalytical methods include, for example, immunoaffinity procedures suchas radioimmunoassay, ELISA or FACS analysis. Analytical methods alsoinclude both in vitro and in vivo imaging procedures. Specific examplesof diagnostic labels that can be detected by analytical means includeenzymes, radioisotopes, fluorochromes, chemiluminescent markers, andbiotin. A label can be attached directly to an antibody of theinvention, fragment thereof, peptide or nucleic acid, or be attached toa secondary binding agent that specifically binds a molecule of theinvention. Such a secondary binding agent can be, for example, asecondary antibody. A secondary antibody can be either polyclonal ormonoclonal, and of human, rodent or chimeric origin.

As described in Example 3, the inventors have demonstrated how arecombinant full length IgG1 human monoclonal antibody of the inventionmay be made. For example, the antibody or functional fragment thereofmay be produced by a bacteriophage expression system. Preferably, thebacteriophage expression system comprises a phage display library.

A useful procedure for isolating the polynucleotide which encodes theantibody or functional fragment thereof begins with isolation of cDNAwhich can be reverse-transcribed from RNA isolated from an individualsuffering from cancer, such as CLL. This disease state is characterizedby the presence of antibodies with immunospecificity against ROR1.Methods for cDNA synthesis are well known in the art. A cDNA encoding anantibody or functional fragment thereof including a heavy or light chaincan be amplified using, for example, the polymerase chain reaction(PCR), preferably reverse transcription PCR (RT-PCR).

Suitable primers for PCR may be determined by those skilled in the artusing conserved sequences which flank the particular functional fragmentof a heavy or light chain. Suitable PCR conditions may be determined bythose skilled in the art.

Preferably, the PCR is adapted to amplify the heavy chain, morepreferably the VH CH1 fragment, and even more preferably, the heavychain variable fragment (VH). Alternatively, or additionally, the PCRmay be adapted to amplify the light chain, more preferably the VL CLfragment, and even more preferably, the light chain variable fragment(VL). Preferably, the PCR products are cloned into a suitable expressionvector, more preferably a phage expression vector, one embodiment ofwhich is illustrated in FIGS. 14a and 14b . Preferably, the vector isintroduced into a suitable host, for example, Chinese hamster ovary(CHO) cells, for expression of the heavy and preferably, the lightfragment, to occur. A suitable vector and host cell system can allow,for example, co-expression and assembly of functional fragments of theheavy and light chains. Preferably, the vector is introduced into thehost by electroporation. Other suitable systems for the expression ofantibody fragments can be determined by those skilled in the art andinclude, for example, M13 phage expression vectors. Recombinantmonoclonal antibodies or functional fragments thereof can besubstantially purified using methods known in the art, and which dependon the particular vector and host expression system used.

In a thirteenth aspect, there is provided a genetic construct comprisingthe nucleic acid of the fourth aspect. Genetic constructs of theinvention may be in the form of an expression cassette, which may besuitable for expression of the encoded polypeptide in a host cell. Thegenetic construct may be introduced in to a host cell without it beingincorporated in a vector. For instance, the genetic construct, which maybe a nucleic acid molecule, may be incorporated within a liposome or avirus particle. Alternatively, a purified nucleic acid molecule (e.g.,histone-free DNA, or naked DNA) may be inserted directly into a hostcell by suitable means, e.g., direct endocytotic uptake. The geneticconstruct may be introduced directly in to cells of a host subject(e.g., a bacterial cell) by transfection, infection, electroporation,microinjection, cell fusion, protoplast fusion or ballistic bombardment.Alternatively, genetic constructs of the invention may be introduceddirectly into a host cell using a particle gun. Alternatively, thegenetic construct may be harbored within a recombinant vector, forexpression in a suitable host cell.

Therefore, in a fourteenth aspect, there is provided a recombinantvector comprising the genetic construct according to the thirteenthaspect.

The recombinant vector may be a plasmid, cosmid or phage. Suchrecombinant vectors are useful for transforming host cells with thegenetic construct of the thirteenth aspect, and for replicating theexpression cassette therein. The skilled technician will appreciate thatgenetic constructs of the invention may be combined with many types ofbackbone vector for expression purposes. Examples of suitable backbonevectors include those shown in FIGS. 14a and 14b . Recombinant vectorsmay include a variety of other functional elements including a suitablepromoter to initiate gene expression. For instance, the recombinantvector may be designed such that it autonomously replicates in thecytosol of the host cell. In this case, elements which induce orregulate DNA replication may be required in the recombinant vector.Alternatively, the recombinant vector may be designed such that itintegrates into the genome of a host cell. In this case, DNA sequenceswhich favor targeted integration (e.g., by homologous recombination) areenvisaged.

The recombinant vector may also comprise DNA coding for a gene that maybe used as a selectable marker in the cloning process, i.e., to enableselection of cells that have been transfected or transformed, and toenable the selection of cells harboring vectors incorporatingheterologous DNA. Alternatively, the selectable marker gene may be in adifferent vector to be used simultaneously with vector containing thegene of interest. The vector may also comprise DNA involved withregulating expression of the coding sequence, or for targeting theexpressed polypeptide to a certain part of the host cell.

In a fifteenth aspect, there is provided a host cell comprising thegenetic construct according to the thirteenth aspect, or the recombinantvector according to the fourteenth aspect.

The host cell may be a bacterial cell. The host cell may be an animalcell, for example a mouse or rat cell. It is preferred that the hostcell is not a human cell. The host cell may be transformed with geneticconstructs or vectors according to the invention, using knowntechniques. Suitable means for introducing the genetic construct intothe host cell will depend on the type of cell. According to a sixteenthaspect, there is provided a method of preparing a recombinant antibodyor functional fragment thereof, the method comprising (i) culturing atleast one cell defined in the fifteenth aspect capable of expressing therequired antibody or functional fragment thereof; and (ii) isolating theantibody or functional fragment thereof.

As described herein, antibodies according to the invention can bind toROR1. However, the inventors realize that the antibody or functionalfragment thereof of the invention may be used to act as a framework forthe development of antibodies showing immunospecificity against othermembers of the ROR family. For example, through introducing mutationsinto the CDRs of the ROR1-specific antibodies described herein, it ispossible to isolate antibodies that can recognize ROR2 in oneembodiment, or both ROR 1 and ROR2 in another embodiment.

Hence, according to a seventeenth aspect, there is provided a method ofisolating an antibody or a functional fragment thereof having ability tobind to Receptor Tyrosine Kinase-Like Orphan Receptor 2 (ROR2), themethod comprising:

-   -   (i) mutating an antibody or functional fragment thereof as        defined in the first aspect to produce a mutant, and    -   (ii) selecting the mutant for immunospecificity against ROR2.

The mutant may be specific for ROR2, or for both ROR1 and ROR2.

In a first embodiment, said mutating step may comprise randommutagenesis, for example using degenerative PCR. For example, cDNA forthe antibody is used as a template in a PCR reaction which may be dopedwith a mutagen, such as a mutagenic nucleoside triphosphate, for exampledP and 8oxo-2′deoxyguanosine. Advantageously, this allows theintroduction of mutations in a highly controlled manner throughout thecDNA to produce a mutant library. The resultant library of mutants maybe displayed on the surface of a phage, and antibodies may then beselected against ROR2.

The resultant library of mutant antibodies may be selected against ROR2using biopanning. For example, an ELISA plate may be coated with ROR2,such as with 100 μl of a 1 pg/ml⁻¹ solution of ROR2 in bicarbonatebuffer pH 8.6, and incubated overnight at 4° C. The plate may then bewashed with buffer, such as TBS. The plate may then be blocked, forexample with 5% BSA in PBS and incubated for one hour at 37° C. Aftertwo further washes, 100 μl phage suspension may be added to each welland the plate may be incubated for two hours at 37° C.

The phage may be removed and the wells filled with TBS 0.05% Tween 20(TBST) and pipetted vigorously. After 5 minutes, the TBST may beremoved, and for a first round of panning, the plate may be washed bythis method once. In a second round of panning, 5 washes may be used,and in a third and subsequent rounds 10 washes may be used. The phagemay then be eluted with 50 pl of elution buffer per well and incubatedat room temperature for 10 minutes. After vigorous pipetting, elutedphage may be removed and neutralized with 3 pl of 2M Tris base.

In a second embodiment, said mutating may comprise introducing at leastone ligand having immunospecificity against ROR2 into at least oneantigen binding region of the antibody of the first aspect. The at leastone ligand may comprise at least one of the six CDRs of a ROR2-specificantibody, which may include:

Light Chain CDR1: (SEQ ID NO: 231) RSSQSLVHSDGNTYLN; Light Chain CDR2:(SEQ ID NO: 232) KVSNRDS; Light Chain CDR3: (SEQ ID NO: 233) MQGTQWPIT;Heavy Chain CDR1: (SEQ ID NO: 234) SYSMN; Heavy Chain CDR2:(SEQ ID NO: 235) YISSSSSTIYYADSVKG;  and Heavy Chain CDR3:(SEQ ID NO: 236) DYGGNSGYYYYYYMDV.

The at least one antigen binding region may be in the heavy and/or lightchain variable fragment. Preferably, the at least one ligand isintroduced into any of the antigen binding regions in the heavy chain ofthe immunoglobulin or functional fragment thereof.

The ligand may be inserted by restriction enzyme digestion at anappropriate site determined by a variety of techniques includingmolecular modelling. A polynucleotide sequence encoding the ligandpeptide sequence may be ligated into the cut restriction site, the exactdetails of this depending on the nature of ligand and the CDR beingused.

According to an eighteenth aspect, there is provided a library or panelof recombinant antibodies or functional fragments thereof, generatedusing the method defined in the seventeenth aspect.

It will be appreciated that the invention extends to any nucleic acid orpeptide or variant, derivative or analogue thereof, which comprisessubstantially the amino acid or nucleic acid sequences of any of thesequences referred to herein, including functional variants orfunctional fragments thereof. The terms “substantially the aminoacid/nucleotide/peptide sequence”, “functional variant” and “functionalfragment”, can be a sequence that has at least 40% sequence identitywith the amino acid/nucleotide/peptide sequences of any one of thesequences referred to herein, for example 40% identity with the sequenceidentified as SEQ ID NO:2 (i.e., the polypeptide sequence of oneembodiment of human ROR1) or the nucleotide identified as SEQ ID NO:1(i.e., the DNA sequence encoding one embodiment of human ROR1), and soon.

Amino acid/polynucleotide/polypeptide sequences with a sequence identitywhich is greater than 50%, more preferably greater than 65%, 70%, 75%,and still more preferably greater than 80% sequence identity to any ofthe sequences referred to are also envisaged. Preferably, the aminoacid/polynucleotide/polypeptide sequence has at least 85% identity withany of the sequences referred to, more preferably at least 90%, 92%,95%, 97%, 98%, and most preferably at least 99% identity with any of thesequences referred to herein.

The skilled technician will appreciate how to calculate the percentageidentity between two amino acid/polynucleotide/polypeptide sequences. Inorder to calculate the percentage identity between two aminoacid/polynucleotide/polypeptide sequences, an alignment of the twosequences must first be prepared, followed by calculation of thesequence identity value. The percentage identity for two sequences maytake different values depending on: (i) the method used to align thesequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman(implemented in different programs), or structural alignment from 3Dcomparison; and (ii) the parameters used by the alignment method, forexample, local vs global alignment, the pair-score matrix used (e.g.,blosum62, pam250, gonnet etc.), and gap-penalty, e.g., functional formand constants.

Having made the alignment, there are many different ways of calculatingpercentage identity between the two sequences. For example, one maydivide the number of identities by: (i) the length of shortest sequence;(ii) the length of alignment; (iii) the mean length of sequence; (iv)the number of non-gap positions; or (iv) the number of equivalencedpositions excluding overhangs. Furthermore, it will be appreciated thatpercentage identity is also strongly length dependent. Therefore, theshorter a pair of sequences is, the higher the sequence identity one mayexpect to occur by chance.

Hence, it will be appreciated that the accurate alignment of protein orDNA sequences is a complex process. The popular multiple alignmentprogram ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22,4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882)is a preferred way for generating multiple alignments of proteins or DNAin accordance with the invention. Suitable parameters for ClustalW maybe as follows: For DNA alignments: Gap Open Penalty=15.0, Gap ExtensionPenalty=6.66, and Matrix=Identity. For protein alignments: Gap OpenPenalty=10.0, Gap Extension Penalty=0.2, and Matrix=Gonnet. For DNA andProtein alignments: ENDGAP=−1, and GAPDIST=4. Those skilled in the artwill be aware that it may be necessary to vary these and otherparameters for optimal sequence alignment.

Preferably, calculation of percentage identities between two aminoacid/polynucleotide/polypeptide sequences may then be calculated fromsuch an alignment as (N/T)*100, where N is the number of positions atwhich the sequences share an identical residue, and T is the totalnumber of positions compared including gaps but excluding overhangshence, a most preferred method for calculating percentage identitybetween two sequences comprises (i) preparing a sequence alignment usingthe clustalw program using a suitable set of parameters, for example, asset out above; and (ii) inserting the values of n and t into thefollowing formula: sequence identity=(N/T)*100.

Alternative methods for identifying similar sequences will be known tothose skilled in the art. For example, a substantially similarnucleotide sequence will be encoded by a sequence which hybridizes toany of the nucleic acid sequences shown herein, or their complementsunder stringent conditions. By stringent conditions, we mean thenucleotide hybridises to filter-bound DNA or RNA in 3× sodiumchloride/sodium citrate (SSC) at approximately 45° C. followed by atleast one wash in 0.2×ssc/0.1% SDS at approximately 20-65° C.Alternatively, a substantially similar polypeptide may differ by atleast 1, but less than 5, 10, 20, 50 or 100 amino acids from thesequences shown herein.

Due to the degeneracy of the genetic code, it is clear that any nucleicacid sequence described herein could be varied or changed withoutsubstantially affecting the sequence of the protein encoded thereby, toprovide a functional variant thereof. Suitable nucleotide variants arethose having a sequence altered by the substitution of different codonsthat encode the same amino acid within the sequence, thus producing asilent change. Other suitable variants are those having homologousnucleotide sequences but comprising all, or portions of, sequence, whichare altered by the substitution of different codons that encode an aminoacid with a side chain of similar biophysical properties to the aminoacid it substitutes, to produce a conservative change. For example smallnon-polar, hydrophobic amino acids include glycine, alanine, leucine,isoleucine, valine, proline, and methionine. Large non-polar,hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.The polar neutral amino acids include serine, threonine, cysteine,asparagine and glutamine. The positively charged (basic) amino acidsinclude lysine, arginine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. It will thereforebe appreciated which amino acids may be replaced with an amino acidhaving similar biophysical properties, and the skilled technician willknow the nucleotide sequences encoding these amino acids.

All of the features described herein (including any accompanying claims,abstract and drawings), and/or all of the steps of any method or processso disclosed, may be combined with any of the above aspects in anycombination, except combinations where at least some of such featuresand/or steps are mutually exclusive.

EXAMPLES Example 1 Generation of Candidates of Therapeutic Human mAbsImmunospecific for Human ROR1 Extracellular Domain (ECD)

Through panning against the human ROR1 extracellular domain (ECD)protein using a fully-human antibody phage library, multipleROR1-specific antibodies were identified that exhibited a wide range ofaffinity (IC50=0.11-263 nM on phage level; see Table 1).

TABLE 1 Affinity ranking of ROR1-positive phage clones by IC50 Clone No.IC50 (nM) P601-1 0.11 P601-40 0.20 P601-109 0.22 P601-43 0.23P601-3(601-3-16)* 0.27 P601-17 0.62 P601-51 0.62 P601-108 0.76 P601-40.93 P601-13 1.21 P601-101 1.50 P601-153 1.53 P601-2 (601-3-12)* 1.75P601-137 1.82 P601-86 1.84 P601-56 1.85 P601-120 1.92 P601-6 2.12P601-18 2.44 P601-134 2.68 P601-81 2.76 P601-149 2.76 P601-112 2.89P601-136 3.02 P601-130 3.08 P601-14 3.22 P601-87 3.86 P601-66 4.05P601-147 4.05 P601-57 4.08 P601-65 4.23 P601-110 4.57 P601-141 5.68P601-100 5.80 P601-5 (601-3-2)* 7.99 P601-28 8.05 P601-103 8.72 P601-699.81 P601-102 10.21 P601-9 14.62 P601-119 17.70 P601-50 24.06 P601-3727.93 P601-70 74.00 P601-128 263.18 *Clones selected to convert intofull-length human IgG1 antibodies

The process is described in the flow chart shown in FIG. 4. A total of1519 single-chain variable fragment (scFv) and fragment antigen-binding(Fab) phage antibodies were screened from enriched panning pools byprotein ELISA and cell ELISA to identify antibodies recognizing nativeconformation of human ROR1 ECD. Using protein ELISA, the inventorsselected over 100 unique clones exhibiting positive binding to bothhuman ROR1 (hROR1) monomeric and dimeric ECD-Fc fusion proteins, andnegative binding to the control Fc-fusion protein and blank control(FIG. 5).

Through cell ELISA, the inventors further identified 45 unique clonesrecognizing ROR1 positive cells, but not ROR1 negative cells (FIG. 6).These 45 cell ELISA-positive phage antibody clones possessing unique DNAcoding sequences (as shown in Table 2) were then subjected to furthercharacterization.

TABLE 2 ROR1 phage antibody display panning summary Cell Type of ELISAUnique ELISA Libraries Planning methods positive rate clone positivescFvnaïve Plastic panning  87/120 15/120  5/120 scFvnaïve Plasticpanning  574/1079 119/1079  38/1079 Panning in solution 24/96 5/96 0/96Cell panning 29/92 4/92 2/92 Fab naïve Plastic panning  83/108 14/108 —Spleen Panning in solution 11/24 2/24 —

Binding affinities of the 45 unique cell ELISA-positive phage cloneswere estimated by competitive phage-binding ELISA. A diagramdemonstrating the design of ROR1 competitive ELISA is shown in FIG. 7.Purified phage antibodies were first diluted serially in PBST buffer,and tested for binding to a ROR1-coated plate. The dilution that gave50-80% saturating signal was used in the solution binding assay in whichphage were first incubated with increasing concentration of ROR1 for oneto two hours, and then transferred to a ROR1-coated plate for 10-15minutes to capture the unbound phage. IC50 was calculated as theconcentration of ROR1 in solution-binding stage that inhibited 50% ofthe phage from binding to immobilized ROR1, and Table 1 summarizes theIC50s of the 45 clones tested.

By epitope grouping assay, the 45 unique phage antibodies were thengrouped into four different classes according to their distinct bindingepitopes on ROR1, as shown in Table 3).

TABLE 3 Epitope bucket summary of ROR1-positive phage clones Total 45Epitope Class I 16 Epitope Class II 10 Epitope Class III 11 EpitopeClass IV 8

In this assay, ROR1 was captured by immobilized anti-ROR1 monoclonalfull-length antibodies with known and distinct binding epitopes. Then,purified phage antibodies were added to the solution. In cases where thebinding between ROR1 and immobilized mAb blocked the binding of thephage antibody to ROR1, this phage antibody was considered to have abinding epitope very close to, or the same as, the immobilized mAb.Otherwise, the phage antibody is thought to have a different epitope.

The complementary determining regions (CDR) regions of both heavy chainand light chain of ROR1-specific antibodies have been analyzed andhighly conserved motifs have been identified within each epitope class(Class I to IV), as illustrated in FIGS. 8-11. FIG. 8 illustratesepitope Class I antibody CDR sequence analysis, FIG. 9 illustratesepitope Class II CDR sequence analysis, FIG. 10 illustrates epitopeClass III CDR sequence analysis, and FIG. 11 illustrates epitope ClassIV CDR sequence analysis.

As can be seen in the figures, there are three subgroups in epitopeClass I, five subgroups in epitope Class II, and two subgroups in eachof epitope classes III and IV. The conserved residues are important fordefining the antibodies' binding specificity and affinity to hROR1-ECD,and provide useful information for future protein engineering to modifythe binding profile.

Binding specificities of the 45 phage antibodies were determined byprotein ELISA against human ROR1-ECD, human ROR2-ECD and mouse ROR1-ECD(FIG. 12). All 45 antibodies show specific binding to ROR1. Thirty-eightout of the 45 antibodies recognize both human and mouse ROR1-ECD (hROR1and mROR1, respectively), as summarized in Table 4, which is a desiredproperty facilitating future preclinical animal studies.

TABLE 4 Binding specificity of ROR1 phage antibodies SpecificityPositive rate hROR1 positive only 7/45 hROR1 and mROR1 positive 38/45 hROR1 and hROR2 positive 0/45

In another panning effort, the inventors discovered clones that bind toboth Ror1 and Ror2, demonstrating that development of ROR1/ROR2bi-specific antibodies is highly feasible (FIG. 13).

Example 2 Summary of Preferred ROR1 Specific Antibodies

The inventors isolated 45 antibodies according to the invention. Theantibodies may be developed for therapeutic or diagnostic use. Fourteenantibodies were selected by the following criteria:

1. They represent all epitope classes and sub-groups;

2. They have high ROR1-binding affinity; and

3. They form the pool that is likely to be converted into full IgGmolecules for further analysis.

For each antibody, the information is organized as following:

1. Name of antibody;

2. Epitope class and sub-group;

3. Light chain variable region DNA sequence;

4. Light chain variable region protein sequence;

5. Light chain CDRs: L1, L2, L3 DNA sequence;

6. Light chain CDRs: L1, L2, L3 amino acid sequence;

7. Heavy chain variable region DNA sequence;

8. Heavy chain variable region protein sequence;

9. Heavy chain CDRs: H1, H2, H3 DNA sequence; and

10. Heavy chain CDRs: H1, H2, H3 amino acid sequence.

1) Antibody 601-1; Epitope Class I; Subgroup 1a 601-1 Light chain variable region (DNA sequence)  [SEQ ID NO: 3]Gccatccggatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccat cacttgccaggcgagtcaggacattagcaactatttaaattggtatcagcagaaaccaggga aagcccctaagctcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggttc agtggaagtggatctgggacagattttactttcaccatcagcagcctgcagcctgaagatat tgcaacatattactgtcaacagtatgataatctccccctcactttcggcggagggaccaagg tggaaatcaaacgt  601-1 Light chain variable region (protein sequence) [SEQ ID NO: 4]AIRMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRF SGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIKR 601-1 Light chain CDRs: L1, L2, L3 (DNA sequence)  [SEQ ID NO: 5]caggcgagtcaggacattagcaactatttaaat  [SEQ ID NO: 6] gatgcatccaatttggaaaca [SEQ ID NO: 7] caacagtatgataatctccccctcact 601-1 Light chain CDRs: L1, L2, L3 (protein sequence)  [SEQ ID NO: 8]QASQDISNYLN  [SEQ ID NO: 9] DASNLET  [SEQ ID NO: 10] QQYDNLPLT 601-1 heavy chain variable region (DNA sequence)  [SEQ ID NO: 11]gaggtgcagctggtggagtctgggggaggcttggtcaagcctggaggatccctgagactctcctgtg cagcctctggattcaccttcagtgactactacatgagctggatccgccaggctccagggaaggggct ggagtgggtttcatacattagtgatagtactaataccatatactacgcagactctgtgaagggccga ttcaccgtctccagggacaaccccaaaaactcactctatctgcaaatgatcagcctgagagccgagg acacggccgtgtattattgtgcgagagctgtgggagctggcgagggctttgaccactggggccaggg aaccctggtcaccgtctcctca 601-1 heavy chain variable region (protein sequence)  [SEQ ID NO: 12]EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISDSTNTIYYAD SVKGRFTVSRDNPKNSLYLQMISLRAEDTAVYYCARAVGAGEGFDHWGQGTLVTVSS 601-1 heavy chain CDRs: H1, H2, H3 (DNA sequence)  [SEQ ID NO: 13]gactactacatgagc  [SEQ ID NO: 14]tacattagtgatagtactaataccatatactacgcagactctgtgaagggc  [SEQ ID NO: 15]gctgtgggagctggcgagggctttgaccac 601-1 heavy chain CDRs: H1, H2, H3 (protein sequence)  [SEQ ID NO: 16]DYYMS  [SEQ ID NO: 17] YISDSTNTIYYADSVKG  [SEQ ID NO: 18] AVGAGEGFDH 2) Antibody 601-5; Epitope Class I; Subgroup 1a 601-5 Light chain variable region (DNA sequence)  [SEQ ID NO: 19]gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccat cacttgccaggcgagtcaggacattagcaactatttaaattggtatcagcagaaaccaggga aagcccctaagctcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggttc agtggaagtggatctgggacagattttaccttcaccatcagcagcctgcagcctgaagatat tgcaacatattactgtcaacagtatgataatctccccctcactttcggcggagggaccaagc tggagatcaaacgt  601-5 Light chain variable region (protein sequence) [SEQ ID NO: 20]DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRF SGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKLEIKR 601-5 Light chain CDRs: L1, L2, L3 (DNA sequence)  [SEQ ID NO: 21]caggcgagtcaggacattagcaactatttaaat  [SEQ ID NO: 22]gatgcatccaatttggaaaca  [SEQ ID NO: 23] caacagtatgataatctccccctcact 601-5 Light chain CDRs: L1, L2, L3 (protein sequence)  [SEQ ID NO: 24]QASQDISNYLN  [SEQ ID NO: 25] DASNLET  [SEQ ID NO: 26] QQYDNLPLT 601-5 heavy chain variable region (DNA sequence)  [SEQ ID NO: 27]gaggtgcagctggtggagtctgggggaggcttggtcaagcctggagggtccctgagactctc ctgtgcagcctctggattcaccttcagtgactactacatgggctgggtccgccaggctccgg ggaagggccttaagtggctttcatacattagtgatcgtgcgcataccatatacgacacagac tctgtgaagggccgattcaccatttccagggacgacgccaagagttcgctttatctgcgaat gaacaacctgagagtcgaggacacggccgtttactactgtgcgagggcagtgggagctgggg agggctttgactactggggccaaggcaccctggtgaccgtctcctca 601-5 heavy chain variable region (protein sequence)  [SEQ ID NO: 28]EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMGWVRQAPGKGLKWLSYISDRAHTIYDTD SVKGRFTISRDDAKSSLYLRMNNLRVEDTAVYYCARAVGAGEGFDYWGQGTLVTVSS 601-5 heavy chain CDRs: H1, H2, H3 (DNA sequence)  [SEQ ID NO: 29]gactactacatgggc  [SEQ ID NO: 30]tacattagtgatcgtgcgcataccatatacgacacagactctgtgaagggc  [SEQ ID NO: 31]gcagtgggagctggggagggctttgactac 601-5 heavy chain CDRs: H1, H2, H3 (protein sequence)  [SEQ ID NO: 32]DYYMG  [SEQ ID NO: 33] YISDRAHTIYDTDSVKG  [SEQ ID NO: 34] AVGAGEGFDY 3) Antibody 601-51; Epitope Class I; Subgroup 1b 601-51 Light chain variable region (DNA sequence)  [SEQ ID NO: 35]cagtctgccctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctc ctgcactggaaccagcagtgactttggtgattatgactatgtctcttggtaccaacaacacc caggcaaagcccccaaactcatgatttatgatgtcagtgatcggccctcaggggtttctaat cgcttctctggctccaagtctggcaacacggcctccctgaccatctctgggctccaggctga ggacgaggctgattatttctgcagctcatttacaaccagcagcactctggtgttcggcggag ggaccaagctgaccgtcctaggt 601-51 Light chain variable region (protein sequence)  [SEQ ID NO: 36]QSALTQPASVSGSPGQSITISCTGTSSDFGDYDYVSWYQQHPGKAPKLMIYDVSDRPSGVSN RFSGSKSGNTASLTISGLQAEDEADYFCSSFTTSSTLVFGGGTKLTVLG 601-51 Light chain CDRs: L1, L2, L3 (DNA sequence)  [SEQ ID NO: 37]actggaaccagcagtgactttggtgattatgactatgtctct  [SEQ ID NO: 38]gatgtcagtgatcggccctca  [SEQ ID NO: 39] agctcatttacaaccagcagcactctggtg 601-51 Light chain CDRs: L1, L2, L3 (protein sequence)  [SEQ ID NO: 40]TGTSSDFGDYDYVS  [SEQ ID NO: 41] DVSDRPS  [SEQ ID NO: 42] SSFTTSSTLV601-51 heavy chain variable region (DNA sequence)  [SEQ ID NO: 43]caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctc ctgcaaggcttctggatacaccttcaccggctactatatgcactgggtgcgacaggcccctg gacaagggcttgagtggatgggatggatcaaccctaacagtggtggcacaaactatgcacag aagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagct gagcaggctgagatctgacgacacggccgtgtattactgtgcgagagatggggatatggtct atgatagtagtgggcctgactactggggccagggaaccctggtcaccgtctcctca 601-51 heavy chain variable region (protein sequence)  [SEQ ID NO: 44]QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQ KFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDGDMVYDSSGPDYWGQGTLVTVSS 601-51 heavy chain CDRs: H1, H2, H3 (DNA sequence)  [SEQ ID NO: 45]Ggctactatatgcac  [SEQ ID NO: 46]Tggatcaaccctaacagtggtggcacaaactatgcacagaagtttcagggc  [SEQ ID NO: 47]Gatggggatatggtctatgatagtagtgggcctgactac 601-51 heavy chain CDRs: H1, H2, H3 (protein sequence)  [SEQ ID NO: 48]GYYMH  [SEQ ID NO: 49] WINPNSGGTNYAQKFQG  [SEQ ID NO: 50] DGDMVYDSSGPDY 4) Antibody 601-4; Epitope Class I; Subgroup 1c 601-4 Light chain variable region (DNA sequence)  [SEQ ID NO: 51]cagtctgtgctgactcagccaccctcagcgtctggggcccccgggcagagggtcaccatctc ctgttccggaggcatctccaacgtcgggactaatggtgttaactggtaccagcacctcccag gaacggcccccaaactcctcgtcgatgctatgaatcagcggccctcaggagtccctgaccga ttctctggctccaggtctggcacgtcaggctccctggccatcactgggctccggtctgaaga tgaggctgactattattgtgcaacatgggatgacagcctgagtggtgtactattcggcggag ggaccaagctgaccgtcctaggt 601-4 Light chain variable region (protein sequence)  [SEQ ID NO: 52]QSVLTQPPSASGAPGQRVTISCSGGISNVGTNGVNWYQHLPGTAPKLLVDAMNQRPSGVPDR FSGSRSGTSGSLAITGLRSEDEADYYCATWDDSLSGVLFGGGTKLTVLG 601-4 Light chain CDRs: L1, L2, L3 (DNA sequence)  [SEQ ID NO: 53]tccggaggcatctccaacgtcgggactaatggtgttaac  [SEQ ID NO: 54]gctatgaatcagcggccctca  [SEQ ID NO: 55] aacatgggatgacagcctgagtggtgtacta 601-4 Light chain CDRs: L1, L2, L3 (protein sequence)  [SEQ ID NO: 56]SGGISNVGTNGVN  [SEQ ID NO: 57] AMNQRPS  [SEQ ID NO: 58] ATWDDSLSGVL 601-4 heavy chain variable region (DNA sequence)  [SEQ ID NO: 59]gaggtgcagctggtgcagtctggcccaggactggtgaagccttcggggaccctgtccctcac ctgcgctgtctctggtggctccatcagcagtagtaactggtggagttgggtccgccagcccc cagggaaggggctggagtggattggggaaatctatcatagtgggagcaccaactacaacccg tccctcaagagtcgagtcaccatatcagtagacaagtccaagaaccagttctccctgaagct gggctctgtgaccgccgcggacacagccacatattactgtgcgcgcgatctgtggctgggtg agtgggatttgtggggccaaggcaccctggtcaccgtctcctca 601-4 heavy chain variable region (protein sequence)  [SEQ ID NO: 60]EVQLVQSGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNP SLKSRVTISVDKSKNQFSLKLGSVTAADTATYYCARDLWLGEWDLWGQGTLVTVSS 601-4 heavy chain CDRs: H1, H2, H3 (DNA sequence)  [SEQ ID NO: 61]agtagtaactggtggagt  [SEQ ID NO: 62]gaaatctatcatagtgggagcaccaactacaacccgtccctcaagagt  [SEQ ID NO: 63]gatctgtggctgggtgagtgggatttg 601-4 heavy chain CDRs: H1, H2, H3 (protein sequence)  [SEQ ID NO: 64]SSNWWS  [SEQ ID NO: 65] EIYHSGSTNYNPSLKS  [SEQ ID NO: 66] DLWLGEWDL 5) Antibody 601-2; Epitope Class II; Subgroup 2a 601-2 Light chain variable region (DNA sequence)  [SEQ ID NO: 67]gaaattgtgttgacgcagtctccagacaccctgtccttgtctccaggggaaagagccaccct ctcctgcagggccagtcagagtgttagcagcaacttagcctggtaccagcagaaacctggcc aggctcccaggctcctcatctatggtgcatccaccagggccactggtatcccagccaggttc agtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattt tgcagtttattactgtcagcagtataataactggcctccgtacacttttggccaggggacca aggtggaaatcaaacgt  601-2 Light chain variable region (protein sequence) [SEQ ID NO: 68]EIVLTQSPDTLSLSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARF SGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPPYTFGQGTKVEIKR 601-2 Light chain CDRs: L1, L2, L3 (DNA sequence)  [SEQ ID NO: 69]agggccagtcagagtgttagcagcaacttagcc  [SEQ ID NO: 70]ggtgcatccaccagggccact  [SEQ ID NO: 71] cagcagtataataactggcctccgtacact 601-2 Light chain CDRs: L1, L2, L3 (protein sequence)  [SEQ ID NO: 72]RASQSVSSNLA  [SEQ ID NO: 73] GASTRAT  [SEQ ID NO: 74] QQYNNWPPYT 601-2 heavy chain variable region (DNA sequence)  [SEQ ID NO: 75]caggtcaccttgaaggagtctgggcccacgctggtgaaacccacacagaccctcacgctgac gtgcaccttctctggcttctcactcagtagttttggagtggctgtgggctggttccgtcagc ccccaggaaaggccctggagtggcttggacttatttattgggatgatgataagcgctacagc ccatctctgaagaccaggctcaccatcaccaaggacacctccaaaaaccaggtggtccttac aatgaccaacatggaccctgtggacacagccacatattattgtgcccacaaagggggtatag caacaactggcagccccaactggttcgacccctggggccagggaaccctggtcaccgtctcc  tca 601-2 heavy chain variable region (protein sequence)  [SEQ ID NO: 76]QVTLKESGPTLVKPTQTLTLTCTFSGFSLSSFGVAVGWFRQPPGKALEWLGLIYWDDDKRYS PSLKTRLTITKDTSKNQVVLTMTNMDPVDTATYYCAHKGGIATTGSPNWFDPWGQGTLVTVS  S 601-2 heavy chain CDRs: H1, H2, H3 (DNA sequence)  [SEQ ID NO: 77]agttttggagtggctgtgggc  [SEQ ID NO: 78]cttatttattgggatgatgataagcgctacagcccatctctgaagacc  [SEQ ID NO: 79]aaagggggtatagcaacaactggcagccccaactggttcgacccc 601-2 heavy chain CDRs: H1, H2, H3 (protein sequence)  [SEQ ID NO: 80]SFGVAVG  [SEQ ID NO: 81] LIYWDDDKRYSPSLKT  [SEQ ID NO: 82]KGGIATTGSPNWFDP  6) Antibody 601-17; Epitope Class II; Subgroup 2b 601-17 Light chain variable region (DNA sequence)  [SEQ ID NO: 83]gaaattgtgttgacgcagtctccaggcaccctgtctttgtctccaggggaaagagccaccct ctcctgcagggccagtcagagtgttagcagcagctacttagcctggtaccagcagaaacctg gccaggctcccaggctcctcatctatggtgcatccagcagggccactggcatcccagacagg ttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaaga ttttgcagtgtattactgtcagcagtatggtagcctttttggccaggggaccaaggtggaga tcaaacgt  601-17 Light chain variable region (protein sequence) [SEQ ID NO: 84]EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLFGQGTKVEIKR 601-17 Light chain CDRs: L1, L2, L3 (DNA sequence)  [SEQ ID NO: 85]agggccagtcagagtgttagcagcagctacttagcct  [SEQ ID NO: 86]ggtgcatccagcagggccact  [SEQ ID NO: 87] cagcagtatggtagcctt 601-17 Light chain CDRs: L1, L2, L3 (protein sequence)  [SEQ ID NO: 88]RASQSVSSSYLA  [SEQ ID NO: 89] GASSRAT  [SEQ ID NO: 90] QQYGSL 601-17 heavy chain variable region (DNA sequence)  [SEQ ID NO: 91]gaggtccagctggtacagtctggggctgaggtgaggaaacctgggtcctcggtgaaggtctc ctgcaaggcctctggaggctccctcagcagtcatggtgtcagttgggtgcgtcaggcccctg gacaagggcttgagtggatggccaggatcatccccatgtttggtctaacagactacgcacag aacttccaggccagagtcacgatttccgcggacagatccacgaacacagtttacatggagat cagcaacctgggatctgaagacacggccgtctatttctgtgcgagagagagtctgggagcaa catttgagtattggggccagggaaccctggtcaccgtctcctca 601-17 heavy chain variable region (protein sequence)  [SEQ ID NO: 92]EVQLVQSGAEVRKPGSSVKVSCKASGGSLSSHGVSWVRQAPGQGLEWMARIIPMFGLTDYAQ NFQARVTISADRSTNTVYMEISNLGSEDTAVYFCARESLGATFEYWGQGTLVTVSS 601-17 heavy chain CDRs: H1, H2, H3 (DNA sequence)  [SEQ ID NO: 93]agtcatggtgtcagt  [SEQ ID NO: 94]aggatcatccccatgtttggtctaacagactacgcacagaacttccaggcc  [SEQ ID NO: 95]gagagtctgggagcaacatttgagtat 601-17 heavy chain CDRs: H1, H2, H3 (protein sequence)  [SEQ ID NO: 96]SHGVS  [SEQ ID NO: 97] RIIPMFGLTDYAQNFQA  [SEQ ID NO: 98] ESLGATFEY 7) Antibody 601-119; Epitope Class II; Subgroup 2c 601-119 Light chain variable region (DNA sequence)  [SEQ ID NO: 99]cagtctgccctgactcagcctgcctccgtgtctgcgtctcctggacagtcgatcaccatctc ctgcactggaaccagcagtgacgttggtggttataactatgtcacctggtaccaacagcacc caggcaaagcccccaaactcatgatttatgatgtcagtaagcggccctcaggggtccttgat cgcttctctggctccaagtctggcaacacggcctccctgaccatctctgggctccaggctga ggacgaggctgattatttctgcagctcatatacaagcagttccaccctggtgtttggcggag ggaccaagctgaccgtcctaggt 601-119 Light chain variable region (protein sequence)  [SEQ ID NO: 100]QSALTQPASVSASPGQSITISCTGTSSDVGGYNYVTWYQQHPGKAPKLMIYDVSKRPSGVLD RFSGSKSGNTASLTISGLQAEDEADYFCSSYTSSSTLVFGGGTKLTVLG 601-119 Light chain CDRs: L1, L2, L3 (DNA sequence)  [SEQ ID NO: 101]actggaaccagcagtgacgttggtggttataactatgtcacc  [SEQ ID NO: 102]gatgtcagtaagcggccctca  [SEQ ID NO: 103] agctcatatacaagcagttccaccctggtg 601-119 Light chain CDRs: L1, L2, L3 (protein sequence) [SEQ ID NO: 104] TGTSSDVGGYNYVT  [SEQ ID NO: 105] DVSKRPS [SEQ ID NO: 106] SSYTSSSTLV 601-119 heavy chain variable region (DNA sequence)  [SEQ ID NO: 107]caggtgcagctggtgcaatctgggggaggcctggtcaagcctggggggtccctgagactctc ctgtgcagcctctggattcaccttcggtacctatagcatgaactgggtccgccaggctccag gaaaggggctggagtgggtctcatccattagtagtagtagtagttacatatactacgcagac tcagtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaat gaacagcctgagagccgaggacacggctgtgtattactgtgcgagaggtctcggtggctgga cccatgatgcttttgatatctggggccaagggaccacggtcaccgtctcctca 601-119 heavy chain variable region (protein sequence)  [SEQ ID NO: 108]QVQLVQSGGGLVKPGGSLRLSCAASGFTFGTYSMNWVRQAPGKGLEWVSSISSSSSYIYYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGLGGWTHDAFDIWGQGTTVTVSS 601-119 heavy chain CDRs: H1, H2, H3 (DNA sequence)  [SEQ ID NO: 109]acctatagcatgaac  [SEQ ID NO: 110]tccattagtagtagtagtagttacatatactacgcagactcagtgaagggc  [SEQ ID NO: 111]ggtctcggtggctggacccatgatgcttttgatatc 601-119 heavy chain CDRs: H1, H2, H3 (protein sequence) [SEQ ID NO: 112] TYSMN  [SEQ ID NO: 113] SISSSSSYIYYADSVKG [SEQ ID NO: 114] GLGGWTHDAFDI 8) Antibody 601-14; Epitope Class II; Subgroup 2d 601-14 Light chain variable region (DNA sequence)  [SEQ ID NO: 115]cagtctgtgctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctc ctgcactggaaccagcagtgacgttggtggttataactatgtctcctggtaccaacaacacc caggcaaagcccccaaactcatgatttatgatgtcagtaatcggccctcaggggtttctaat cgcttctctggctccaagtctggcaacacggcctccctgaccatctctgggctccaggctga ggacgaggctgattattactgcagctcatatacaagcagcagcactctttgggtgttcggcg gagggaccaagctgaccgtcctaggt 601-14 Light chain variable region (protein sequence)  [SEQ ID NO: 116]QSVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSN RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLWVFGGGTKLTVLG 601-14 Light chain CDRs: L1, L2, L3 (DNA sequence)  [SEQ ID NO: 117]actggaaccagcagtgacgttggtggttataactatgtctcc  [SEQ ID NO: 118]gatgtcagtaatcggccctca  [SEQ ID NO: 119]agctcatatacaagcagcagcactctttgggtg 601-14 Light chain CDRs: L1, L2, L3 (protein sequence)  [SEQ ID NO: 120]TGTSSDVGGYNYVS  [SEQ ID NO: 121] DVSNRPS  [SEQ ID NO: 122] SSYTSSSTLWV 601-14 heavy chain variable region (DNA sequence)  [SEQ ID NO: 123]caggtgcagctggtgcagtctggagcagaggtgaaaaagcccggggagtctctgaagatctc ctgtaaggattctggatacagctttaccaactactggctcggctgggtgcgccagatgcccg ggaaaggcctggagtggatgggaatcatctatccgggtgactctgataccagatacagcccg tccttccgaggccaggtcaccatctcagccgacaagtccatcagcaccgcctacctgcagtg gagcagcctgaaggcctcggacaccgccatgtattactgtgcgagacttaatcttgccacac atacagcttttgacatatggggccaagggaccacggtcaccgtctcctca 601-14 heavy chain variable region (protein sequence)  [SEQ ID NO: 124]QVQLVQSGAEVKKPGESLKISCKDSGYSFTNYWLGWVRQMPGKGLEWMGIIYPGDSDTRYSP SFRGQVTISADKSISTAYLQWSSLKASDTAMYYCARLNLATHTAFDIWGQGTTVTVSS 601-14 heavy chain CDRs: H1, H2, H3 (DNA sequence)  [SEQ ID NO: 125]aactactggctcggc  [SEQ ID NO: 126]atcatctatccgggtgactctgataccagatacagcccgtccttccgaggc  [SEQ ID NO: 127]cttaatcttgccacacatacagcttttgacata 601-14 heavy chain CDRs: H1, H2, H3 (protein sequence)  [SEQ ID NO: 128]NYWLG  [SEQ ID NO: 129] IIYPGDSDTRYSPSFRG  [SEQ ID NO: 130] LNLATHTAFDI 9) Antibody 601-86; Epitope Class II; Subgroup 2e 601-86 Light chain variable region (DNA sequence)  [SEQ ID NO: 131]gacatccagatgacccagtctccatcttctgtgtctgcatctgtaggagacagagtcaccat cacttgtcgggcgagtcaaggtattagcaccttgttggcctggtatcagcagaaaccaggga aagcccctaagctcctgatatcttctgcatccagtttgcaaagtggggtcccagcaaggttc agcggcagtggatctgggacagatttcactctcactatcagcagcctgcagcctgaggattt tgcaacttactactgccagcaaagttacagagccccgactttcggccaggggaccaaggtgg agatcaaacgt  601-86 Light chain variable region (protein sequence) [SEQ ID NO: 132]DIQMTQSPSSVSASVGDRVTITCRASQGISTLLAWYQQKPGKAPKLLISSASSLQSGVPARF SGSGSGTDFTLTISSLQPEDFATYYCQQSYRAPTFGQGTKVEIKR 601-86 Light chain CDRs: L1, L2, L3 (DNA sequence)  [SEQ ID NO: 133]cgggcgagtcaaggtattagcaccttgttggcc  [SEQ ID NO: 134]tctgcatccagtttgcaaagt  [SEQ ID NO: 135] cagcaaagttacagagccccgact 601-86 Light chain CDRs: L1, L2, L3 (protein sequence)  [SEQ ID NO: 136]RASQGISTLLA  [SEQ ID NO: 137] SASSLQS  [SEQ ID NO: 138] QQSYRAPT 601-86 heavy chain variable region (DNA sequence)  [SEQ ID NO: 139]caggtcaccttgaaggagtctggtcctacgctgctgaaacccacacagaccctcacgctgac ctgcaccttctctgggttctcactcagtactagaggagtgggggtgggctggatccgtcagc ccccaggacaggccctggagtggcttacactcatttattgggatgatgataagcgctacagc ccttctctaaagagcaggctcaccatcaccaaggacacatccaaaaaccaggtggtccttac aatgaccaacatggaatctgtggacacagccacatattactgtgcacagcagactatgaccg gtgcttttgatatctggggccaagggaccacggtcaccgtctcctca 601-86 heavy chain variable region (protein sequence)  [SEQ ID NO: 140]QVTLKESGPTLLKPTQTLTLTCTFSGFSLSTRGVGVGWIRQPPGQALEWLTLIYWDDDKRYS PSLKSRLTITKDTSKNQVVLTMTNMESVDTATYYCAQQTMTGAFDIWGQGTTVTVSS 601-86 heavy chain CDRs: H1, H2, H3 (DNA sequence)  [SEQ ID NO: 141]actagaggagtgggggtgggc  [SEQ ID NO: 142]ctcatttattgggatgatgataagcgctacagcccttctctaaagagc  [SEQ ID NO: 143]cagactatgaccggtgcttttgatatc 601-86 heavy chain CDRs: H1, H2, H3 (protein sequence)  [SEQ ID NO: 144]TRGVGVG  [SEQ ID NO: 145] LIYWDDDKRYSPSLKS  [SEQ ID NO: 146] QTMTGAFDI 10) Antibody 601-40; Epitope Class III; Subgroup 3a 601-40 Light chain variable region (DNA sequence)  [SEQ ID NO: 147]gacatccagttgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccat cacttgccgggcaagtcagaacattaacaactatttaaattggtatcagcagaaaccaggga aagcccctaagctcctgctctatgctgcatccagtttgcaaagtggggtcccatcaaggttc agtggcagtggatctgggacagaattcactctcaccatcagcagtctgcaccctgaagattt tgcaacttactactgtcaacagagttacaataccccattcaccttcggccctgggaccaaag tggatatcaaacgt  601-40 Light chain variable region (protein sequence) [SEQ ID NO: 148]DIQLTQSPSSLSASVGDRVTITCRASQNINNYLNWYQQKPGKAPKLLLYAASSLQSGVPSRF SGSGSGTE  FTLTISSLHPEDFATYYCQQSYNTPFTFGPGTKVDIKR 601-40 Light chain CDRs: L1, L2, L3 (DNA sequence)  [SEQ ID NO: 149]cgggcaagtcagaacattaacaactatttaaat  [SEQ ID NO: 150]gctgcatccagtttgcaaagt  [SEQ ID NO: 151] caacagagttacaataccccattcacc 601-40 Light chain CDRs: L1, L2, L3 (protein sequence)  [SEQ ID NO: 152]RASQNINNYLN  [SEQ ID NO: 153] AASSLQS  [SEQ ID NO: 154] QQSYNTPFT 601-40 heavy chain variable region (DNA sequence)  [SEQ ID NO: 155]caggtacagctgcagcagtcaggagctgaggtgaagaagcctggggcctcagtgaaggtctc ctgcaaggcttctggttacacctctaccaactatggtatcagctgggtgcgacaggcccctg gacaagggcttgagtggatgggatggatcagcacttacaatggtaacacaaactatgcacag aagctccagggcagagtcaccatgaccacagacacatccacgagcacagcctacatggagct gaggagcctgagatctgacgacacggccgtgtattactgtgcgagagactattactctgata gtagtggttattgggacgatgcttttgatatctggggccaagggacaatggtcaccgtctct  tca 601-40 heavy chain variable region (protein sequence)  [SEQ ID NO: 156]QVQLQQSGAEVKKPGASVKVSCKASGYTSTNYGISWVRQAPGQGLEWMGWISTYNGNTNYAQ KLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDYYSDSSGYWDDAFDIWGQGTMVTVS  S 601-40 heavy chain CDRs: H1, H2, H3 (DNA sequence)  [SEQ ID NO: 157]aactatggtatcagc  [SEQ ID NO: 158]tggatcagcacttacaatggtaacacaaactatgcacagaagctccagggc  [SEQ ID NO: 159]gactattactctgatagtagtggttattgggacgatgcttttgatatc 601-40 heavy chain CDRs: H1, H2, H3 (protein sequence)  [SEQ ID NO: 160]NYGIS  [SEQ ID NO:161] WISTYNGNTNYAQKLQG  [SEQ ID NO: 162]DYYSDSSGYWDDAFDI  11) Antibody 601-13; Epitope Class III; Subgroup 3b 601-13 Light chain variable region (DNA sequence)  [SEQ ID NO: 163]gacatcgtgatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccat cacttgccaggcgagtcaggacattagcaactatttaaattggtatcagcagaaaccaggga aagcccctaagctcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggttc agtggaagtggatctgggacagattttattttcaccatcagcagcctgcagcctgaagatat tgcaacatattactgtcaacagtttgataatctcccttacacttttggccaggggaccaagg tggagatcaaacgt  601-13 Light chain variable region (protein sequence) [SEQ ID NO: 164]DIVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRF SGSGSGTDFIFTISSLQPEDIATYYCQQFDNLPYTFGQGTKVEIKR 601-13 Light chain CDRs: L1, L2, L3 (DNA sequence)  [SEQ ID NO: 165]caggcgagtcaggacattagcaactatttaaat  [SEQ ID NO: 166]gatgcatccaatttggaaaca  [SEQ ID NO: 167] caacagtttgataatctcccttacact 601-13 Light chain CDRs: L1, L2, L3 (protein sequence)  [SEQ ID NO: 168]QASQDISNYLN  [SEQ ID NO: 169] DASNLET  [SEQ ID NO: 170] QQFDNLPYT 601-13 heavy chain variable region (DNA sequence)  [SEQ ID NO: 171]gaggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctc ctgcaaggcttctggtggcaccttcagcacctttgcgatcaactgggtgcgacaggcccctg gacaagggcttgagtggatgggaggggtcatccctgtctctggaacagaagactactcacag aagttccagggcagactctcacttaccgcggacgagtccacgggcacagcctacatggagct gagcagcctgagatctgacgacacggccgtgtattactgtgcgagagatcgaagtggccgcg attgggactactttgactattggggccagggaaccctggtcaccgtctcctca 601-13 heavy chain variable region (protein sequence)  [SEQ ID NO: 172]EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTFAINWVRQAPGQGLEWMGGVIPVSGTEDYSQ KFQGRLSLTADESTGTAYMELSSLRSDDTAVYYCARDRSGRDWDYFDYWGQGTLVTVSS 601-13 heavy CDRs: H1, H2, H3 (DNA sequence)  [SEQ ID NO: 173]acctttgcgatcaac  [SEQ ID NO: 174]ggggtcatccctgtctctggaacagaagactactcacagaagttccagggc  [SEQ ID NO: 175]gatcgaagtggccgcgattgggactactttgactat 601-13 heavy CDRs: H1, H2, H3 (protein sequence)  [SEQ ID NO: 176]TFAIN  [SEQ ID NO: 177] GVIPVSGTEDYSQKFQG  [SEQ ID NO: 178]DRSGRDWDYFDY  12) Antibody 601-109; Epitope Class IV; Subgroup 4a 601-109 Light chain variable region (DNA sequence)  [SEQ ID NO: 179]caatctgccctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctc ctgcactggaaccagcagtgacgttggtggttataactatgtctcctggtaccaacagcacc caggcaaagcccccaaactcttgatttatgaggtcagtcagcggccctcaggggtccctgat cgattctctggctccaagtctggcaacacggcctccctgaccgtctctggcctccaggctga agatgaggctgactattattgcagctcatatgcaggcgacagggacgtcttcggaactggga cccagctcaccgttttaagt 601-109 Light chain variable region (protein sequence)  [SEQ ID NO: 180]QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLLIYEVSQRPSGVPD RFSGSKSGNTASLTVSGLQAEDEADYYCSSYAGDRDVFGTGTQLTVLS 601-109 Light chain CDRs: L1, L2, L3 (DNA sequence)  [SEQ ID NO: 181]actggaaccagcagtgacgttggtggttataactatgtctcc  [SEQ ID NO: 182]gaggtcagtcagcggccctca  [SEQ ID NO: 183] agctcatatgcaggcgacagggacgtc 601-109 Light chain CDRs: L1, L2, L3 (protein sequence) [SEQ ID NO: 184] TGTSSDVGGYNYVS  [SEQ ID NO: 185] EVSQRPS [SEQ ID NO: 186] SSYAGDRDV 601-109 heavy chain variable region (DNA sequence)  [SEQ ID NO: 187]cagatgcagctggtgcagtctgggggagacttggtccagcctggggggtccctgagactctc ctgtgcagcctctggattcaccttcagtagctatagcatgaactgggtccgccaggctccag gaaaggggctggagtgggtctcatccattagtagtagtagtagttacatatactacgcagac tcagtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaat gaacagcctgagagccgaggacacggctgtgtattactgtgcgagaggtctcggtggctgga cccatgatgcttttgatatctggggccaagggaccacggtcaccgtctcctca 601-109 heavy chain variable region (protein sequence)  [SEQ ID NO: 188]QMQLVQSGGDLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYAD SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGLGGWTHDAFDIWGQGTTVTVSS 601-109 heavy chain CDRs: H1, H2, H3 (DNA sequence)  [SEQ ID NO: 189]agctatagcatgaac  [SEQ ID NO: 190]tccattagtagtagtagtagttacatatactacgcagactcagtgaagggc  [SEQ ID NO: 191]ggtctcggtggctggacccatgatgcttttgatatc 601-109 heavy chain CDRs: H1, H2, H3 (protein sequence) [SEQ ID NO: 192] SYSMN  [SEQ ID NO: 193] SISSSSSYIYYADSVKG [SEQ ID NO: 194] GLGGWTHDAFDI 13) Antibody 601-137; Epitope Class IV; Subgroup 4b 601-137 Light chain variable region (DNA sequence)  [SEQ ID NO: 195]cagtctgccctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctc ttgttctggaagcagctccaacatcggaagtaattatgtatactggtaccagcagctcccag gaacggcccccaaactcctcatctataggaataatcagcggccctcaggggtccctgaccga ttctctggctccaagtctggcacctcagcctccctggccatcagtgggctccggtccgagga tgaggctgattattactgtgcagcatgggatgacagcctgagtgcctgggtgttcggcggag ggaccaagctgaccgtcctaggt 601-137 Light chain variable region (protein sequence)  [SEQ ID NO: 196]QSALTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDR FSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSAWVFGGGTKLTVLG 601-137 Light chain CDRs: L1, L2, L3 (DNA sequence)  [SEQ ID NO: 197]tctggaagcagctccaacatcggaagtaattatgtatac  [SEQ ID NO: 198]aggaataatcagcggccctca  [SEQ ID NO: 199]gcagcatgggatgacagcctgagtgcctgggtg 601-137 Light chain CDRs: L1, L2, L3 (protein sequence) [SEQ ID NO: 200] SGSSSNIGSNYVY  [SEQ ID NO: 201] RNNQRPS [SEQ ID NO: 202] AAWDDSLSAWV 601-137 heavy chain variable region (DNA sequence)  [SEQ ID NO: 203]caggtacagctgcagcagtcaggggctgaggtgaagaagcctggggcctcagtgaaggtttc ctgcaaggcatctggatacaccttctccagatactatatccactgggtgcgacaggcccctg gtcaagggcttgagtggatgggaataatcaacactgatggtggcaccacaacctacgcacag aagtttcagggcagactcaccatgaccagggacacgtccacgagcaccgtctacatggaact gagcagcctgagatctgacgacacggccgtctattactgtgcgagagattatgggactatag atgctcgtcgttttgactactggggccagggaaccctggtcaccgtctcctca 601-137 heavy chain variable region (protein sequence)  [SEQ ID NO: 204]QVQLQQSGAEVKKPGASVKVSCKASGYTFSRYYIHWVRQAPGQGLEWMGIINTDGGTTTYAQ KFQGRLTMTRDTSTSTVYMELSSLRSDDTAVYYCARDYGTIDARRFDYWGQGTLVTVSS 601-137 heavy chain CDRs: H1, H2, H3 (DNA sequence)  [SEQ ID NO: 205]agatactatatccac  [SEQ ID NO: 206]ataatcaacactgatggtggcaccacaacctacgcacagaagtttcagggc  [SEQ ID NO: 207]gattatgggactatagatgctcgtcgttttgactac 601-137 heavy chain CDRs: H1, H2, H3 (protein sequence) [SEQ ID NO: 208] RYYIH  [SEQ ID NO: 209] IINTDGGTTTYAQKFQG [SEQ ID NO: 210] DYGTIDARRFDY 14) Antibody 601-3; Epitope Class II; Subgroup 2a 601-3 Light chain variable region (DNA sequence)  [SEQ ID NO: 211]gaaatagtgatgacgcagtccccagccaccctgtctgtgtctccaggggaaagagccaccct ctcctgcagggccagtcagagtgttagcagcaacttagcctggtaccagcagaaacctggcc aggctcccaggctcctcatctatggtgcatccaccagggccactggtatcccagccaggttc agtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattt tgcagtttattactgtcagcagtataataactggcctccgtacacttttggccaggggacca aggtggagatcaaacgt  601-3 Light chain variable region (protein sequence) [SEQ ID NO: 212]EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARF SGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPPYTFGQGTKVEIKR 601-3 Light chain CDRs: L1, L2, L3 (DNA sequence)  [SEQ ID NO: 213]agggccagtcagagtgttagcagcaacttagcc  [SEQ ID NO: 214]ggtgcatccaccagggccact  [SEQ ID NO: 215] cagcagtataataactggcctccgtacact 601-3 Light chain CDRs: L1, L2, L3 (protein sequence)  [SEQ ID NO: 216]RASQSVSSNLA  [SEQ ID NO: 217] GASTRAT  [SEQ ID NO: 218] QQYNNWPPYT 601-3 heavy chain variable region (DNA sequence)  [SEQ ID NO: 219]caggtcaccttgaaggagtctgggcccacgctggtgaaacccacacagaccctcacgctgac gtgcaccttctctggcttctcactcaatagttttggagtggctgtgggctggttccgtcagc ccccaggaaaggccctggagtggcttggacttatttattgggatgatgacaggcgctacttc ccatcgctggagggcaggctctccatcaccaaggacgcctccgataacaacgtggtcctgac aatgatgaacgtggaccctgcggacacagccacatattattgtgcacggacttcccctatgg ttcagggaattgcaaactactacgctatggacgtctggggccaagggaccacggtcaccgtc  tcctca 601-3 heavy chain variable region (protein sequence)  [SEQ ID NO: 220]QVTLKESGPTLVKPTQTLTLTCTFSGFSLNSFGVAVGWFRQPPGKALEWLGLIYWDDDRRYF PSLEGRLSITKDASDNNVVLTMMNVDPADTATYYCARTSPMVQGIANYYAMDVWGQGTTVTV  SS 601-3 heavy chain CDRs: H1, H2, H3 (DNA sequence)  [SEQ ID NO: 221]agttttggagtggctgtgggc  [SEQ ID NO: 222]cttatttattgggatgatgacaggcgctacttcccatcgctggagggc  [SEQ ID NO: 223]acttcccctatggttcagggaattgcaaactactacgctatggacgtc 601-3 heavy chain CDRs: H1, H2, H3 (protein sequence)  [SEQ ID NO: 224]SFGVAVG  [SEQ ID NO: 225] LIYWDDDRRYFPSLEG  [SEQ ID NO: 226]TSPMVQGIANYYAMDV The inventors have also determined the amino acid and DNA sequences for the constant regions of one embodiment of the antibody,as follows.  Antibody light chain constant region (protein sequence) [SEQ ID NO: 227]EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARF SGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPPYTFGQGTKVEIKR Antibody heavy chain constant region (protein sequence) [SEQ ID NO: 228]QVTLKESGPTLVKPTQTLTLTCTFSGFSLNSFGVAVGWFRQPPGKALEWLGLIYWDDDRRYF PSLEGRLSITKDASDNNVVLTMMNVDPADTATYYCARTSPMVQGIANYYAMDVWGQGTTVTV  SS Antibody light chain constant region (DNA sequence)  [SEQ ID NO: 229]gaaatagtgatgacgcagtccccagccaccctgtctgtgtctccaggggaaagagccaccct ctcctgcagggccagtcagagtgttagcagcaacttagcctggtaccagcagaaacctggcc aggctcccaggctcctcatctatggtgcatccaccagggccactggtatcccagccaggttc agtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattt tgcagtttattactgtcagcagtataataactggcctccgtacacttttggccaggggacca aggtggagatcaaacgt  Antibody heavy chain constant region (DNA sequence) [SEQ ID NO: 230]caggtcaccttgaaggagtctggacccacgctggtgaaacccacacagaccctcacgctgac gtgcaccttctctggcttctcactcaatagttttggagtggctgtgggctggttccgtcagc ccccaggaaaggccctggagtggcttggacttatttattgggatgatgacaggcgctacttc ccatcgctggagggcaggctctccatcaccaaggacgcctccgataacaacgtggtcctgac aatgatgaacgtggaccctgcggacacagccacatattattgtgcacggacttcccctatgg ttcagggaattgcaaactactacgctatggacgtctggggccaagggaccacggtcaccgtc  tcctca 

Example 3 Engineering Full Length Monoclonal Antibodies Using theSelected scFv Fragments

Although the phage display technology allows for the rapid selection andproduction of antigen-specific scFv fragments, the complete monoclonalantibodies (mAbs) including the Fc domains have a number of advantagesover the scFv. Firstly, only full lengths antibodies exert immunologicalfunction such as complement-dependent cytotoxicity (CDC) andantibody-dependent cytotoxicity (ADCC) mediated via the Fc domain.Secondly, bivalent monoclonal antibodies offer stronger antigen-bindingaffinity than monomeric Fab Abs. Thirdly, plasma half-life and renalclearance will be different with the Fab and bivalent mAb. Fourthly,bivalent mAb may be internalized at different rates from scFv, alteringimmune function or carrier function. Alpha emitters do not need to beinternalized to kill the targets, but many drugs and toxins will benefitfrom internalization of the immune complex. Therefore, according to theaffinity ranking result obtained through competitive ELISA (see Table1), three clones with high binding affinity were then selected andreconstructed into full-length human IgG1 recombinant antibodies andcharacterized further.

To produce recombinant human monoclonal IgG in Chinese hamster ovary(CHO) cells, the inventors engineered full length IgG1 mAbs based on themethod by Tomomatsu et al., 2009, Biosci Biotechnol Biochem 73(7):1465-1469. Antibody variable regions were sub-cloned into mammalianexpression vectors (FIGS. 14a and 14b ), with matching Kappa light chainconstant sequences and IgG1 subclass Fc (FIG. 15). Purified full-lengthIgG1 antibodies showed expected molecular weights under both reducingand non-reducing conditions (FIG. 16). Kinetic binding analysisconfirmed specific binding of the full length IgG1 antibodies tohROR1-ECD, with a Kd in picomolar range (FIG. 17).

Example 4 Characterization of the ROR1-Specific Full Length IgG1Monoclonal Antibodies

1. Epitope Mapping:

To better understand the structural basis of the ROR1-specific antibodyas a drug candidate, a standard epitope mapping was performed by ELISAagainst a ROR1 peptide array. The peptide array covered the whole lengthof the ROR1 protein extracellular domain with 96 overlapping peptides(15 amino acids each with a 5 amino acid overlap). The peptides werebiotinylated at the N-terminal to immobilize them onto streptavidinELISA plates. A spacer (SGSG) was used to provide flexibility. ELISAassays were performed with the ROR1 antibodies as the primary antibodyand an anti-human Fc AP-conjugated antibody was used for detection.

As shown in FIGS. 18, 19 and 20, antibody 601-3-12 (FIG. 19) and601-3-16 (FIG. 20) share similar binding epitopes, while antibody601-3-2 (FIG. 18) has distinct binding epitopes different from 601-3-12and 601-3-16.

2. Binding Property:

Binding specificity was tested again using the three purified fulllength IgG1 antibodies. Protein ELISA confirmed that they all recognizeboth human and mouse ROR1 ECDs, but not human ROR2 ECD (FIG. 21).

Binding of the hIgG1 mAbs to the cell surface ROR1 was determined byFACS analysis using different cancer cell lines positive for ROR1expression, including MDA-MB-231, A549, H1299 and Jeko-1. All threeantibodies recognize ROR1 expressed on the surface of cancer cells. Anexample of FACS using MDA-MB-231 cells is shown in FIG. 22. Theantibodies at 0.1 ug/ml, 1 ug/ml, 10 ug/ml and 100 ug/ml were incubatedwith MDA-MB-231 cells, then with FITC-labeled secondary antibody afterwashing. The binding was measured by FACS and expressed as meanfluorescence intensity (MFI). Cells incubated with secondary antibodyonly were used as negative control. FIG. 22 shows the results of 10ug/ml ROR1-specific mAbs.

3. Immune Activity:

The inventors then evaluated in vitro immune effector mechanisms of theROR1-specific IgG1 mAbs, such as CDC and ADCC:

A) Complement-Dependent Cytotoxicity (CDC) Activity

An important mechanism by which mAbs kill cells is that antibody boundto target cell surface fixes complement, which results in initiation ofthe complement cascade and assembly of the membrane attack complex (MAC)that ultimately results in lysis of the target cell. The inventorsinvestigated whether the ROR1-specific IgG mAbs were capable ofattracting Clq binding, the first step of initiating complement complexformation. Various concentrations of ROR1 antibodies were coated ontoplate, and 2 ug/mL of the Clq protein was used as a probe. Horse RadishProtein (HRP)-conjugated anti-Clq antibody was used to report thebinding signal. As shown in FIG. 23, all three antibodies were able tobind to Clq in a dose-dependent manner, proving that the antibodies canmediate complement-dependent cytotoxicity (CDC) of the target cells.

B) Antibody-Dependent Cellular Cytotoxicity (ADCC) Against ROR1 PositiveCancer Cell Lines

Antibody-dependent cellular cytotoxicity is one of the importanteffector mechanisms of mAb therapy. The antibody can bridge tumor cellsexpressing the target molecules and cytotoxic cells such as NK cells,and mediate NK-directed target cell killing. The ADCC assay wasperformed as follows. 100 ul of target cell suspension (MDA-MB-231 orJeko-1) were pre-incubated with 50 ul of the testing mAbs at the variousconcentrations in 96-well plate at 37° C. for half hour. 50 ul offreshly isolated human peripheral blood mononuclear cells (PBMC) werethen added at the effector/target cell ratio of 25:1. After incubatedfor 16 hours, the plate was spun down and 50 ul of cell-freesupernatants were transferred to a new plate. The released LDH fromkilled cancer cells was measured by CytoTox96 Non-radioactiveCytotoxicity Assay from Promega. The cell lysis was calculated by theformula (E-S)/(M-S) (E: experimental release, S: spontaneous release, M:maximal release). Non-specific antibody was used as a negative control.The inventors found that all three antibodies were capable of mediatingkilling of ROR1-expressing tumor cells in the ADCC assay. FIG. 24 showsthe results of ADCC against MDA-MB-231 cells by ROR1-specific mAbs.

4. Blockade of Wnt5a Ligand Binding:

Furthermore, the three mAbs were tested in three biological assays toevaluate their functional activities:

-   -   (i) Blocking of Wnt5a binding to ROR1; and    -   (ii) Blocking of Wnt-5a induced ROR1 phosphorylation

A ForteBio assay was designed to test the ability of the three antibodycandidates to block Wnt-5a binding to ROR1. ROR1/Wnt5A interaction wasmeasured using ForteBio Octet. Three anti-ROR1 mAbs and one hIgG1negative control antibody M901 were then added, respectively, to measurethe reduction of binding (measured a Pseudo ka (1/M.s). Compared withthe negative control IgG1 antibody, all the three antibodies were ableto block Wnt-5a binding to ROR1 to various extents (FIG. 25).

Upon binding to ROR1, Wnt5a triggers ROR1 phosphorylation and downstreamsignaling cascade activation. ROR1 phosphorylation leads to the slowermigration of Ror1 on SDS-PAGE electrophoresis. To determine if theirlead candidate antibodies against ROR1 could functionally block Wnt5abinding to ROR1, they made use of this assay as the surrogate experimentto test this possibility. They incubated serum-starved ROR1-expressingcell line Jeko-1 with 300 nM Wnt5a treated with or without 10 lag of theantibodies for 2 hours. The cells were lysed, separated by SDS-PAGEelectrophoresis, and blotted with antibody against ROR1. Treating thecells with Wnt5a alone caused slower migration of ROR1 when compared tothe non-phosphorylation.

However, the effect of Wnt5a on ROR1 migration was completely abrogatedby two out of three lead candidates (clone 3-12 and clone 3-16) whenboth Wnt5a and the antibodies were co-incubated with the cells. Theresults demonstrate that these antibodies can effectively prevent Wnt5afrom binding to ROR1, and that this blockade of the ligand bindingabolishes ROR1 activation.

5. Inhibition of Cancer Cell Proliferation

Cell proliferation assays were performed to determine if the antibodiesinhibit cancer cell proliferation. Target cells (MDA-MB-231 or SKBR3cells) were plated into 96-well plate at 5×10³ cells per well. 10 ug/mlROR1-specific mAbs were added to the cells and incubated at 37° C. in a5% CO2 incubator. After 72-h treatment, cells were assayed forproliferation using CellTiter 96 AQueous One Solution Cell ProliferationAssay (Promega) according to the manufacturer's instructions. Untreatedcell viability was set as 100% and data was shown as percentage comparedto untreated cells. A non-specific antibody (901) was used as a negativecontrol. Each data point represents mean±S.D. (n=3).

Example 5 Exploration of Further Improvement of ROR1 AntibodyDruggability

1. Conjugation to Cytotoxic Moieties

1) Antibody Drug Conjugate

Antibody-drug conjugate (ADC) is used for an antibody to deliver apotent cytotoxic drug selectively to a target cell. Such methods, whenapplied to a tumor antigen target, can enhance the antitumor activity ofantibodies and improve the tumor-to-normal tissue selectivity ofchemotherapy. One key parameter for ADC development is that the antibodycan be endocytosed once bound to target antigen, and therefore, deliverthe conjugated drug into target cancer cells.

To explore the possibility of developing ADC strategy with the ROR1antibodies of the invention, the inventors evaluated endocytosis of theantibodies induced by ROR1-antibody binding. In the assay, Jeko cellswere surface-labeled for 60 min on ice with FITC-labeled ROR1 antibodies(10 ug/ml) in PBS with 5% FBS, and then washed three times with cold PBSand incubated at 37° C. in RPMI with 5% FBS for 3 hrs to internalizesurface fluorescence. Cells were rapidly chilled, washed and incubatedwith (stripped) or without (non-stripped) 500 ul stripping buffer (150mM NaCl+HCL, pH 2.5) for 5 min at RT, spin down, wash twice with PBS andthen fixed with cold 1% PFA/PBS and immediately analyzed by flowcytometry. Internalized fluorescence was calculated from stripped andnon-stripped sample data after correcting from incomplete surfacestripping.

As shown in FIG. 26, the inventors found that ROR1 antibodies wereslowly internalized.

2) Conjugation to Alpha Emitting Radionucleotides

Alpha particles are high energy, high linear energy transfer heliumnuclei capable of strong, yet selective single cell cytotoxicity.Targetable nanogenerators are ²²⁵Ac-labeled monoclonal antibodies thathave been demonstrated to be extremely potent and specific in killingleukemia, lymphoma, and other solid tumors (McDevitt et al., 2001,Science 294 (5546): 1537-1540). The inventors decided to conjugate theROR1 antibodies to alpha particle emitters with bi-functional chelates.Given their exquisite specificity for the ROR1, full-length mAb andScFv/Fab can be excellent vehicles for delivering potent anti-tumorreagents. Their small size, rapid clearance from blood and tumorpenetration properties should make ScFv/Fab an ideal format of choicefor tumor-targeted alpha emitters. The inventors conjugated all forms ofmAbs (full length IgG, scFV and Fab) to alpha particle emitters usingthe two-step labeling method as described in Borchardt et al., 2003,Cancer Res 63: 5-84-5090.

3) Test the In Vitro Cytotoxicity of the Conjugates Against ROR1Positive Cancer Cell Lines

A panel of ROR1 positive cell lines was treated with or withoutconjugated ROR1 IgG antibodies ranging from 0.01-10 ug/ml and negativecontrol antibody conjugates for 24-96 hours. Cell proliferation wasdetermined by 3H-thymidine incorporation, and viability by ATPlite assay(Griffiths, et al., 2011, Methods Mol Biol 731: 451-65 Sun, et al.,2005, Oncogene 24: 7381-88).

4) Test the In Vivo Anti-Tumor Activity of the Conjugates in a NOD/SCIDMouse Bearing Cancer Cells Expressing ROR1.

A) Therapeutic Efficacy of the Constructs on NOD/SCID Xenograft Model

The NOD/SCID mice are characterized by a functional deficit in NK cells,an absence of circulating complement and defects in the differentiationand function of APCs (antigen-presenting cells). Therefore, the NOD/SCIDmodel is suitable for xenografts of human tumor cell lines and to testthe direct effects of mAb against tumor cells expressing targetedmolecules. A similar protocol was used to establish the mouse model andto perform therapeutic studies to determine the therapeutic efficacy ofthe conjugated mAbs (Francisco, et al., 2003, Blood 102:1458-1465).

B) Bio-Distribution of the Conjugates

This was measured by labeling both forms of mAbs with ¹¹¹In, andcounting the radioactivity from various tissues from mice at differenttime points, after injection (Singh Jaggi et al., 2007, Plos One 2(3):e267). Based on the results obtained from the normal mice, the tumoruptake of the constructs was determined using the NOD/SCID xenograft ofhuman leukemia, by gamma imaging as described previously.

5) Engineer Novel Forms of the Antibodies to Enhance their CytotoxicAbilities

1. Bispecific Antibody

Bispecific antibodies were constructed to recognize both ROR1 and CD3 onimmune T cells as described (Gunasekaran et al., 2010, J. Biol. Chem.285: 19637-19646; Rossi E A et al., 2006, Proc Natl Aca Sci USA103:6841-6) with a human IgG1 Fc. Bispecific antibodies were expected torecruit and target cytotoxic T cells to ROR1 positive cancer cells,while maintaining Fc effector functions and long half-life in vivo.Three mechanisms are involved in the specific killing of cancer cellsmediated by bispecific antibodies: (i) killing by activated T cells;(ii) ADCC activity; (iii) CDC activity. Other formats of bispecificantibodies can be constructed, such tandem scFv molecules (taFv),diabodies (Db), or single chain diabodies (scDb), and fusion proteinwith human serum albumin (Asano et al., J. Biol. Chem. 286: 1812-1818;Loffler et al., Blood 95(6): 2098-2103; Weiner et al., J. Immunology152(5): 2385-2392; Muller et al., J. Biol. Chem. 282: 12650-12660), butare devoid of Fc effector functions with distinct pharmacokineticprofiles.

2. ADCC Enhancement

ROR1 target specific ADCC activity was enhanced by expressing antibodiesrecombinantly in glycol-engineered CHO cells as described inPCT/US2010/0081195. The modified oligosaccharide N-glycan on Asn297alters effector functions as follows: higher affinity binding toCD16/FcRIIIa for improved ADCC activity mediated by human Natural Killercells; 2) reduced binding affinity to CD32b/FcRIIb, an inhibitoryreceptor expressed in multiple types of immune cells (except NK cells),for improved ADCC activity mediated by effector cells such asneutrophils and antigen presentation by macrophage and DC cells.

Example 6 Anti-ROR1 Antibody Binding Affinity

The binding affinities of ROR1 extracellular domain dimer and monomerfor anti-ROR1 antibodies were determined by surface plasmon resonance(SPR). ROR1 extracellular domain Fc-fusion, “dimer,” and ROR1extracellular domain Fc fusion:Fc heterodimer, “monomer,” are the dimerand monomeric forms of ROR1 used for affinity analysis, respectively.Briefly, ROR1 dimer and monomer were directly immobilized to a CM4 chipusing an amine coupling kit and 100 mM ethylenediamine in 100 mM SodiumBorate buffer, pH 8.0 as the blocking reagent. Approximately 100-150 RUof ROR1 dimer and monomer were immobilized on separate flow cells and anun-derivatized flow cell was used as a reference control. Each anti-ROR1antibody was diluted in HBS-P+ running buffer and injected at 5 or 6concentrations (0 nM, 1.23 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM) at30 μl/min for 120 seconds. Dissociation was followed for 180 seconds.The association constant, dissociation constant, and affinity of eachanti-ROR1 antibody was calculated using the Biacore T200 EvaluationSoftware 1:1 binding model.

The binding studies demonstrated that 19 anti-ROR1 antibodies bind toboth ROR1 monomer and dimer with similar affinity when fit with the 1:1binding model, as shown in Table 5. While the interaction betweenanti-ROR1 and ROR1-Fc fusion dimer could be analyzed with the bivalentanalyte, analysis was performed using the 1:1 binding model as bindingcurves for both ROR1 monomer and dimer are similar. Apparent affinityfor anti-ROR1 antibody binding to ROR1 dimer is shown. FIG. 27 showsnormalized sensorgrams of antibodies binding to both ROR1 monomer anddimer. Sensorgrams were normalized to 100 RU using the “bindingstability point” at the end of the association phase but before thedissociation phase.

TABLE 5 ROR1 extracellular domain monomer and dimer binding kinetics andaffinity to anti-ROR1 antibodies by SPR Anti-ROR1 Ab ROR1 analyte ka(1/Ms) kd (1/s) KD (nM)* 601-13 dimer 1.6E+06 8.7E−04 0.5 monomer1.8E+06 1.4E−03 0.8 601-14 dimer 2.7E+06 6.6E−04 0.2 monomer 3.2E+061.3E−03 0.4 601-17 dimer 1.8E+06 9.0E−04 0.5 monomer 2.3E+06 1.0E−03 0.4601-18 dimer 4.9E+06 1.5E−03 0.3 monomer 6.0E+06 2.2E−03 0.4 601-100dimer 4.4E+05 1.7E−03 3.9 monomer 3.5E+05 2.8E−03 8.2 601-103 dimer1.7E+05 3.5E−03 20.7 monomer 1.2E+05 5.0E−03 40.2 601-112 dimer 3.4E+053.2E−04 1.0 monomer 2.7E+05 1.2E−03 4.6 601-119 dimer 4.4E+05 7.9E−02178.0 monomer 2.3E+05 9.5E−02 407.3 601-128A dimer 4.1E+05 2.1E−03 5.0monomer 3.4E+05 2.1E−03 6.3 601-128B dimer 4.3E+06 2.7E−03 0.6 monomer3.6E+06 2.9E−03 0.8 601-130 dimer 5.2E+05 1.2E−03 2.2 monomer 4.5E+051.8E−03 4.1 601-134 dimer 1.8E+05 1.2E−03 6.7 monomer 3.2E+05 4.8E−0314.9 601-14 dimer 2.4E+06 1.5E−03 0.6 monomer 2.4E+06 2.1E−03 0.8601-141 dimer 1.8E+06 2.2E−02 11.9 monomer 1.2E+06 2.6E−02 20.6 601-147dimer 6.8E+05 6.3E−04 0.9 monomer 5.9E+05 1.7E−03 2.8 601-149 dimer1.9E+06 2.3E−04 0.1 monomer 1.6E+06 6.9E−04 0.4 601-28 dimer 3.4E+059.7E−03 28.5 monomer 3.2E+05 1.2E−02 36.4 601-37 dimer 1.0E+06 1.8E−031.7 monomer 9.2E+05 2.7E−03 3.0 601-4 dimer 3.6E+06 7.6E−04 0.2 monomer4.6E+06 1.6E−03 0.3 601-5 dimer 2.5E+06 2.9E−04 0.1 monomer 2.3E+068.4E−04 0.4 601-50 dimer 9.4E+05 1.1E−03 1.1 monomer 1.1E+06 1.6E−03 1.5601-65 dimer 1.1E+06 7.2E−03 6.4 monomer 1.7E+06 1.1E−02 6.6 601-66dimer 3.1E+06 5.1E−03 1.7 monomer 2.5E+06 5.9E−03 2.4 601-70 dimer5.1E+06 1.8E−02 3.5 monomer 3.6E+06 1.9E−02 5.4 601-87 dimer 7.6E+055.8E−04 0.8 monomer 6.0E+05 1.3E−03 2.2 601-9 dimer 1.4E+06 2.1E−02 15.3monomer 6.3E+05 1.7E−02 27.6 *Apparent affinity is reported for ROR1dimer binding to antibody

Example 7 Anti-ROR1 Antibody Binding to CLL Cells

The ability of the antibodies to bind to ROR1 on the surface of primarychronic lymphocytic leukemia (CLL) cells was determined by flowcytometry. ROR1 is not expressed on normal, healthy adult blood cells,but is found on malignant cells of virtually all cases of CLL and ondifferent types of solid tumors. Thirty-seven of the antibodies testedshowed detectable binding to CLL cells by flow cytometry.

Methods Cell Surface Staining of CLL Cells by the CD5, CD19, andAnti-ROR1 Antibodies

Anti-ROR1 and control IgG1 antibodies were labeled using a Zenon® AlexaFluor® 647 human IgG labeling kit (Life Technologies) according to themanufacturer's directions. Frozen peripheral blood mononuclear cells(PBMC) from CLL patients (AllCells LLC) were thawed and washed in FACSbuffer (PBS, 2% heat inactivated FBS, 0.1% NaN3). CLL cells were countedand resuspended at 4×10E6 cell/mL in FACS buffer. 2×10E5 cells per testwere then incubated in 100 pt FACS buffer with phycoerythrin(PE)-labeled anti-CD5, flu601-5cein isothiocyanate (FITC)-labeledanti-CD19 (BD Biosciences) and Zenon® Alexa Fluor® 647-labelledanti-ROR1 or control human IgG1 ET901 (Eureka Therapeutics) for 30minutes on ice. Following incubation, the cells were washed twice inFACS buffer and fixed in 2% paraformaldehyde in PBS (finalconcentration). Cells were run on a BD LSRII flow cytometer (BDBiosciences) and analyzed using FlowJo software (Treestar).

CLL cells identified as CD5 and CD19 double positive were assessed foranti-ROR1 or control Ig staining. FIGS. 28 and 29 show the geometricmean fluorescence intensity (gMFI) of various concentrations of theanti-ROR1 antibody on CLL cells.

Summary

The invention is directed to a human ROR1-specific antibody.Advantageously, the antibody is human in origin, and is therefore likelyto minimize any immune response upon administration to a human patientin contrast to using antibodies comprising non-human elements. Inaddition, the antibody or functional fragment thereof, amino acidsequences and nucleotide sequences encoding the antibody or functionalfragment thereof, and conjugates described herein, may be effectivelyused for the manufacture of compositions and diagnostics, and usesthereof, for example, in treating cancer.

TABLE 6 Certain Light Chain Variable Region Sequences SEQ ID kappa/Antibody Epitope NO: lambda name class Sequence   4 kappa AntibodyEpitope AIRMTQSPSS LSASVGDRVT ITCQASQDIS 601-1 Class INYLNWYQQKP GKAPKLLIYD ASNLETGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCQQYDNLPLTFGG GTKVEIKR  68 kappa Antibody EpitopeEIVLTQSPDT LSLSPGERAT LSCRASQSVS 601-2(3- Class IISNLAWYQQKP GQAPRLLIYG ASTRATGIPA 12) RFSGSGSGTE FTLTISSLQS EDFAVYYCQQYNNWPPYTFG QGTKVEIKR 212 kappa Antibody EpitopeEIVMTQSPAT LSVSPGERAT LSCRASQSVS 601-3(3- Class IISNLAWYQQKP GQAPRLLIYG ASTRATGIPA 16) RFSGSGSGTE FTLTISSLQS EDFAVYYCQQYNNWPPYTFG QGTKVEIKR  52 lambda Antibody EpitopeQSVLTQPPSA SGAPGQRVTI SCSGGISNVG 601-4 Class ITNGVNWYQHL PGTAPKLLVD AMNQRPSGVP DRFSGSRSGT SGSLAITGLR SEDEADYYCATWDDSLSGVL FGGGTKLTVL G  20 kappa Antibody EpitopeDIQMTQSPSS LSASVGDRVT ITCQASQDIS 601-5(3- Class INYLNWYQQKP GKAPKLLIYD ASNLETGVPS 2) RFSGSGSGTD FTFTISSLQP EDIATYYCQQYDNLPLTFGG GTKLEIKR 241 kappa Antibody EpitopeDIQMTQSPSS LSASVGDRVT ITCQASQDIR 601-6 Class INYLNWYQQKP GKAPKLLIYA ASNLETGVPS RFSGSGSGTD FTFTISSLQP EDIATYYCQQDDNLPLTFGG GTKLEIKR 242 lambda Antibody EpitopeQSVLTQPPSV SVAPGETARI TCGGTNIGSE 601-9 Class IIISVHWYQQRPG QAPVLVVYDD TDRPSGIPER FSGSNSGNTA TLTISRVEAG DGADYYCQVWDSVSDRYVFG TGTKVTVLG 164 kappa Antibody EpitopeDIVMTQSPSS LSASVGDRVT ITCQASQDIS 601-13 Class IIINYLNWYQQKP GKAPKLLIYD ASNLETGVPS RFSGSGSGTD FIFTISSLQP EDIATYYCQQFDNLPYTFGQ GTKVEIKR 116 lambda Antibody EpitopeQSVLTQPASV SGSPGQSITI SCTGTSSDVG 601-14 Class IIGYNYVSWYQQ HPGKAPKLMI YDVSNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYCSSYTSSSTLW VFGGGTKLTV LG  84 kappa Antibody EpitopeEIVLTQSPGT LSLSPGERAT LSCRASQSVS 601-17 Class IISSYLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQQYGSLFGQGT KVEIKR 243 kappa Antibody EpitopeEIVLTQSPGT LSLSPGERAT LSCRASQSVS 601-18 Class IISSYLAWYQQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQQYGSSFGPGT KVDIKR 244 lambda Antibody EpitopeQSALTQPASV SGSPGQSITI SCTGTSSDVG 601-28 Class IVGYNYVSWYQQ HPGKAPKLMI FDVSNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYCSSYTSSSTLF GGGTKLTVLG 245 kappa Antibody EpitopeAIRMTQSPSS LSASVGDRVT ITCQASQDIS 601-37 Class INYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQSYSTPFTFGP GTKVDIKR 148 kappa Antibody EpitopeDIQLTQSPSS LSASVGDRVT ITCRASQNIN 601-40 Class IIINYLNWYQQKP GKAPKLLLYA ASSLQSGVPS RFSGSGSGTE FTLTISSLHP EDFATYYCQQSYNTPFTFGP GTKVDIKR 246 kappa Antibody EpitopeDIQLTQSPSS LSASVGDRVT ITCRASQSIS 601-43 Class IIISYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQSYSTPPWTFG QGTKVEIKR 247 lambda Antibody EpitopeQSALTQPPSA SGTPGQRVTI SCPGSSSNIG 601-50 Class IVSNYVYWYQQL PGTAPKLLIY RNNQRPSGVP DRFSGSKSGT SATLGITGLQ TGDEADYYCGTWDSSLSAYV FGTGTKVTVLG  36 lambda Antibody EpitopeQSALTQPASV SGSPGQSITI SCTGTSSDFG 601-51 Class IDYDYVSWYQQ HPGKAPKLMI YDVSDRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYFCSSFTTSSTLV FGGGTKLTVLG 248 lambda Antibody EpitopeQSVLTQPASV SGSPGQSITI SCTGTSSDFG 601-56 Class IDYDYVSWYQQ HPGKAPKLMI YDVSDRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYFCSSLTTSSTLV FGGGTKLTVLG 249 kappa Antibody EpitopeAIQMTQSPSS LSASVGDRVT ITCRTSQSIS 601-57 Class IIISYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQSYSTPFTFGP GTKVDIKR 250 lambda Antibody EpitopeQSVLTQPPSV SGTPGQRVTI FCSGGSNNIG 601-65 Class IIIRSSVYWYRQA AGTAPKLLIY KTDQRPSGVP DRFAASKSGA SASLAISGLR SEDEADYHCATWDDSLSAVV FGGGTKLTVLG 251 lambda Antibody EpitopeQSALTQPALT QPASVSGSPG QSITISCTGT 601-66 Class ISSDFGDYDYV SWYQQHPGKA PKLMIYDVSD RPSGVSNRFS GSKSGNTASL TISGLQAEDEADYFCSSFTT SSTLVFGGGTKLTVLG 252 lambda Antibody EpitopeQSALTQPASV SGSPGQSITI SCTGTSSDVG 601-69 Class IIGYNYVSWYQQ HPGKAPKLMI YDVSNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYCSSYTSSSIPW VFGGGTKLTVLG 253 lambda Antibody EpitopeQSALTQPALT QPASVSGSPG QSITISCTGT 601-70 Class ISSDFGDYDYV SWYQQHPGKA PKLMIYDVSD RPSGVSNRFS GSKSGNTASL TISGLQAEDEADYFCSSFTT SSTLVFGGGTKLTVLG 254 lambda Antibody EpitopeNFMLTQPRSV SESPGKTVTI SCTGNGGRVA 601-81 Class INNYVQWYQQR PGSAPTTVIY EDNQRPSGVP ARFSGSIDSS SNSASLTISG LKTDDEADYYCQSYDISNQR VFGGGTKLTVLG 132 kappa Antibody EpitopeDIQMTQSPSS VSASVGDRVT ITCRASQGIS 601-86 Class IITLLAWYQQKP GKAPKLLISS ASSLQSGVPA RFSGSGSGTD FTLTISSLQP EDFATYYCQQSYRAPTFGQG TKVEIKR 255 kappa Antibody EpitopeETTLTQSPAF MSATPGDKVN ISCKASQDID 601-87 Class IDDLNWYQQKP GEAPILIIQE ATTLVPGIPP RFSGSGFGTD FTLTINSMQS EDVAYYFCLQHDNFPPTFGQ GTKVEIKR 256 lambda Antibody EpitopeQSALTQPASV SGSPGQSITI SCTGTSRDVG 601-100 Class IVGYDYVSWYQQ YPGNAPKLMI YDVSRRPSGV SHRFSASKSG NTASLTISGL QTEDEADYYCSSYTSSSTRV FGGGTKVTVLG 257 kappa Antibody EpitopeDIQLAQSPSS LSASVGDRVT ITCRASQSIS 601-101 Class INYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQSYSTPFTFGP GTKVEIKR 258 lambda Antibody EpitopeQSALTQPASV SGSPGQSITI SCTGTSSDVG 601-102 Class IIGYNYVSWYQQ HPGKAPKLMI YDVSKRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYCSSYTSSSTSV VFGGGTKLTVLG 259 lambda Antibody EpitopeQSVLTQPASV SGSPGQSITI SCTGTTSDVG 601-103 Class IVGYNYVSWYQQ HPGKAPKLMI YDVSKRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYCSSYTSSSTDV FGTGTKLTVLG 260 lambda Antibody EpitopeQSVVTQPPSV SAAPGQKVTI SCSGSSSNIG 601-108 Class IKNYVSWYQQF PGTAPKLLIY DNNERPSGIP ARFSGSKSGT SATLGITGLQ TGDEADYYCATFDTSLWAAV FGGGTKLTVLG 180 lambda Antibody EpitopeQSALTQPASV SGSPGQSITI SCTGTSSDVG 601-109 Class IVGYNYVSWYQQ HPGKAPKLLI YEVSQRPSGV PDRFSGSKSG NTASLTVSGL QAEDEADYYCSSYAGDRDVF GTGTQLTVLS 261 lambda Antibody EpitopeQSALTQPASV SGSPGQSITL SCTGTSSDVG 601-110 Class IIGYNYVSWYQQ HPGNGPKLII YDVTKRPSGV SNRFSGSKSG NTAYLTISGL QAEDEADYYCASYTRSTTLV FGGGTKLTVLG 262 lambda Antibody EpitopeQSALTQPASV SGSPGQSITI SCTGTSSDVG 601-112 Class IVGYNYVSWYQQ HPGKAPKLMI YDVSKRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYCSSYTGRSTVF GGGTKLTVLG 100 lambda Antibody EpitopeQSALTQPASV SASPGQSITI SCTGTSSDVG 601-119 Class IIGYNYVTWYQQ HPGKAPKLMI YDVSKRPSGV LDRFSGSKSG NTASLTISGL QAEDEADYFCSSYTSSSTLV FGGGTKLTVLG 263 lambda Antibody EpitopeQSALTQPASV SGSPGQSITI SCTGTSSDVG 601-120 Class IVGYNYVSWYQQ HPGKAPKLMI YDVSNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYCSSYTSSSTRV FGGGTKLTVLG 264 kappa Antibody EpitopeDIQMTQSPSS VSASVGDRVT ITCRASQGIS 601-128 Class IVSWLAWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQANSFPLTFGG GTKVEIKR 265 lambda Antibody EpitopeQSVLTQPPSV SGAPGQRVTI SCTGSSSNIG 601-130 Class IAGYDVHWYQQ LPGTAPKLLI YGNSNRPSGV PDRFSGSKSG TSASLAITGL QAEDEADYYCQSYDSSLSGY VFGTGTKLTV LG 266 lambda Antibody EpitopeSSELTQDPAV SVALGQTVRI TCQGDSLRSY 601-134 Class IYASWYQQKPG QAPVLVIYGK NNRPSGIPDR FSGSSSGNTA SLTITGAQAE DEADYYCNSRDSSGNHLVFG GGTKLTVLG 267 lambda Antibody EpitopeQSVLTQPPSA SGTPGQRVTI SCSGSSSNIG 601-136 Class IVSNTVNWYQQL PGTAPKLLIY SNNQRPSGVP DRFSGSKSGT SASLAIRGLQ SDDEAEYYCAAWDDSLKSFV FGKGTKVTVLG 196 lambda Antibody EpitopeQSALTQPPSA SGTPGQRVTI SCSGSSSNIG 601-137 Class IVSNYVYWYQQL PGTAPKLLIY RNNQRPSGVP DRFSGSKSGT SASLAISGLR SEDEADYYCAAWDDSLSAWV FGGGTKLTVLG 268 kappa Antibody EpitopeAIQMTQSPSS LSASVGDRVT ITCRASQSIS 601-141 Class IIISHLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQSYSTPFTFGP GTKVDIKR 269 lambda Antibody EpitopeQSALTQPPSA SGTPGQRVTI SCSGSSSNIG 601-147 Class IIISNYVYWYQQL PGTAPKLLIY RNNQRPSGVP DRFSGSKSGT SASLAISGLR SEDEADYYCAAWDDSLSGYV FGTGTKVTVLG 270 kappa Antibody EpitopeDIQLTQSPSS LSASVGDRVT ITCQASQDVR 601-149 Class INYLNWYQQKP GKAPNLLIYD ATNLESGVPS RFSGSGSGTD FTFTISSLQP EDFATYYCQQYDNLPLSFGG GTKVEIKR 271 lambda Antibody EpitopeQSALTQPASV SGSPGQSITI SCTGTSSDVG 601-153 Class IIIGYNYVSWYQQ HPGKAPKFMI YDVSKRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYCGSFTSSITYV FGTGTKVTVL G

TABLE 7 Certain Heavy Chain Variable Region Sequences SEQ ID Epitope NO:Antibody Class Sequence  12 Antibody EpitopeEVQLVESGGG LVKPGGSLRL SCAASGFTFSDYYMSWIRQA 601-1 Class IPGKGLEWVSY ISDSTNTIYY ADSVKGRFTVSRDNPKNSLYLQMISLRAED TAVYYCARAV GAGEGFDHWGQGTLVTVSS  76 Antibody EpitopeQVTLKESGPT LVKPTQTLTL TCTFSGFSLSSFGVAVGWFR 601-2 (3- Class IIQPPGKALEWL GLIYWDDDKR YSPSLKTRLTITKDTSKNQV 12)VLTMTNMDPV DTATYYCAHK GGIATTGSPNWFDPWGQGTL VTVSS 220 Antibody EpitopeQVTLKESGPT LVKPTQTLTL TCTFSGFSLNSFGVAVGWFR 601-3 (3- Class IIQPPGKALEWL GLIYWDDDRR YFPSLEGRLSITKDASDNNV 16)VLTMMNVDPA DTATYYCART SPMVQGIANYYAMDVWGQGT TVTVSS  60 Antibody EpitopeEVQLVQSGPG LVKPSGTLSL TCAVSGGSISSSNWWSWVRQ 601-4 Class IPPGKGLEWIG EIYHSGSTNY NPSLKSRVTISVDKSKNQFSLKLGSVTAAD TATYYCARDL WLGEWDLWGQGTLVTVSS  28 Antibody EpitopeEVQLVESGGG LVKPGGSLRL SCAASGFTFSDYYMGWVRQA 601-5 (3- Class IPGKGLKWLSY ISDRAHTIYD TDSVKGRFTISRDDAKSSLY 2)LRMNNLRVED TAVYYCARAV GAGEGFDYWGQGTLVTVSS 272 Antibody EpitopeEVQLVESGGG LVKPGGSLKL SCAASGFTFSDYYMGWVRQA 601-6 Class IPGKGLKWLSY ISDRAHTIYD THSVKGRFTISRDDAKSSLYLRMNNLRVED TAVYYCARAV GAGEGFDYWCQGTLVTVSS 273 Antibody EpitopeQLQLQESGPG VVKPSGTLSL TCTVSGGSISRSDGYWGWVR 601-9 Class IIIQPPGKGLEWI GSIYDTGTTY YSPSLKSRLIISVDTSKNQFSLTLNSVTAA DTAVYYCASM GGLRSSSSDAFHTWGPGTMV TVSS 172 Antibody EpitopeEVQLVQSGAE VKKPGSSVKV SCKASGGTFSTFAINWVRQA 601-13 Class IIIPGQGLEWMGG VIPVSGTEDY SQKFQGRLSLTADESTGTAYMELSSLRSDD TAVYYCARDR SGRDWDYFDYWGQGTLVTVSS 124 Antibody EpitopeQVQLVQSGAE VKKPGESLKI SCKDSGYSFTNYWLGWVRQM 601-14 Class IIPGKGLEWMGI IYPGDSDTRY SPSFRGQVTISADKSISTAYLQWSSLKASD TAMYYCARLN LATHTAFDIWGQGTTVTVSS  92 Antibody EpitopeEVQLVQSGAE VRKPGSSVKV SCKASGGSLSSHGVSWVRQA 601-17 Class IIPGQGLEWMAR IIPMFGLTDY AQNFQARVTISADRSTNTVYMEISNLGSED TAVYFCARES LGATFEYWGQGTLVTVSS 274 Antibody EpitopeQVQLVQSGTE VKKPGSSVKV SCQASGGSLSSHGVSWLRQA 601-18 Class IIPGQGLEWVGR IIPMFGVTDY AQKFQDRVTITADKSTSTVYMELISLGSDD TAVYFCARES RGATFEYWGQGTLVTVSS 275 Antibody EpitopeEVQLVQSGGD LVQPGGSLRL SCAASGFTFSSYSMNWVRQA 601-28 Class IVPGKGLEWVSS ISSSSSYIYY ADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARGL GGWTHDAFDIWGQGTTVTVSS 276 Antibody EpitopeEVQLVQSGAE VKKPGASVKV SCKASGYTFNNYGFSWVRQA 601-37 Class IPGQGLEWMGW ISVYNGNTNY AQKLQGRVTMTTDTSTSTAYMELRSLRSDD TAVYYCARDY YSDSSGYWDDAFDIWGQGTM VTVSS 156 Antibody EpitopeQVQLQQSGAE VKKPGASVKV SCKASGYTSTNYGISWVRQA 601-40 Class IIIPGQGLEWMGW ISTYNGNTNY AQKLQGRVTMTTDTSTSTAYMELRSLRSDD TAVYYCARDY YSDSSGYWDDAFDIWGQGTM VTVSS 277 Antibody EpitopeQVQLQQSGAE VKKPGASVRV SCKASGYSFGNNGITWVRQA 601-43 Class IIIPGQGLEWMGW ISTYNGNTNY AQKLQGRVTMTTDTSTSTAYMELRSLRSDD TAVYYCARDY YSDSSGYWDDAFDIWGQGTT VTVSS 278 Antibody EpitopeQVQLQQSGAE VKKPGASVKV SCKASGYTFSRYYIHWVRRA 601-50 Class IVPGQGLEWMGL INPGGGSTNY AQKFQGRVTMTRDTSTNTVYLELSSLRSDD TAVYYCARDY GTIDARRFDFWGQGTLVTVSS  44 Antibody EpitopeQVQLVQSGAE VKKPGASVKV SCKASGYTFTGYYMHWVRQA 601-51 Class IPGQGLEWMGW INPNSGGTNY AQKFQGRVTMTRDTSISTAYMELSRLRSDD TAVYYCARDG DMVYDSSGPDYWGQGTLVTV SS 279 Antibody EpitopeQLQLQESGPG LVKPSETLSL TCTVSGGSISSSSYYWGWIR 601-56 Class IQPPGKGLEWI GSIYYSGSTY YNPSLKSRVTISVDTSKNQFSLKLGSVTAA DTAVYYCARH DGTDAFDIWGQGTTVTVSS 280 Antibody EpitopeEVQLVQSGAE VKKPGSSVKI SCKASGGAFTNFGISWVRQA 601-57 Class IIIPGQGLEWMGW ISTYNSETNY AQKLQGRVTMTTDTSTSTAYMELRSLRSDD TAVYYCARDY YSDSSGYWDDAFDIWGQGTL VTVSS 281 Antibody EpitopeQVQLQQSGPG LVKPSQTLSL TCAISGDSVSSNSATWNWIR 601-65 Class IIIQSPSRGLEWL GRTYYRSKWY NDYAVSVKSRIIINPDTSKNQFSLQLNSVT PEDTAVYYCA RGVRAFDIWGQGTTVTVSS 282 Antibody EpitopeQVQLVQSGAE VKEPGASVKV SCKASGYTFRNSGITWVRQA 601-66 Class IPGQGLEWMGW INPNSGGAMY VDNFQGRATMTRDTSINTAYMELRSLSSDD TAVYYCARGM ADLIDVFDIWGQGTLVTVSS 283 Antibody EpitopeQVQLVQSGAE VKKPGESLKI SCKGSGYSFTSYWIGWVRQM 601-69 Class IIPGKGLEWMGI IYPGDSDTRY SPSFQGQVTISADKSISTAYLQWSSLKASD TAMYYCARLS SSSYDAFDIWGQGTMVTVSS 284 Antibody EpitopeEVQLVQSGAE LKKPGSSVRV SCKTSGGSFKTHGISWVRQA 601-70 Class IPGQGLEWMGW INPNSGGALY VDNFQGRATMTRDTSINTAYMELRSLSSDD TAVYYCARGM ADLIDVFDIWGQGTMVTVSS 285 Antibody EpitopeEVQLVESGGG VVRPGGSLRL SCATSGFNFDNYGLSWVRQG 601-81 Class IPGKGLEWMGF IYKSVNTNYS PSLKSRLTISMDTSKNQFSLNLASVTTADT AIYYCARGKV ETSVVDYWGQGTLVTVSS 140 Antibody EpitopeQVTLKESGPT LLKPTQTLTL TCTFSGFSLSTRGVGVGWIR 601-86 Class IIQPPGQALEWL TLIYWDDDKR YSPSLKSRLTITKDTSKNQVVLTMTNMESV DTATYYCAQQ TMTGAFDIWGQGTTVTVSS 286 Antibody EpitopeQVQLQESGPG LVKSSETLSL TCTVSGGSMNNYYWSWIRQP 601-87 Class IAGKGLEWMGR IYSSGSTNYN PALKSRVTMSVDTSKNQFSLNLSSVTAADT AIYYCARASW SGTYWALFDYWGQGTLVTVSS 287 Antibody EpitopeEVQLVQSGGG VVQPGGPLRL SCAASGFTFSSYSMNWVRQA 601-100 Class IVPGKGLEWVSS ISSSSSYIYY ADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARGL GGWTHDAFDIWGQGTMVTVSS 288 Antibody EpitopeEVQLVQSGNE VKRPGASVKV SCKASGHSFS TYGFSWVRQA 601-101 Class IPGQGLEWMGW ISTYNGNTNY AQKLQGRVTM TTDTSTSTAYMELRSLRSDD TAVYYCARDY YSDSSGYWDD AFDIWGQGTL VTVSS 289 Antibody EpitopeQMQLVQSGGD LVQPGGSLRL SCAASGFTFSSYSMNWVRQA 601-102 Class IIPGKGLEWVSS ISSSSSYIYY ADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARGL GGWTHDAFDIWGQGTTVTVS S 290 Antibody EpitopeEVQLVQSGGD LVQPGGSLRL SCAASGFTFSSYSMNWVRQA 601-103 Class IVPGKGLEWVSS ISSSSSYIYY ADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARGL GGWTHDAFDIWGQGTTVTVS S 291 Antibody EpitopeQLQLQESGPG LVKPSETLSL TCTVSGGSISSNSYYWGWIR 601-108 Class IQPPGKGLEWI GSIYYSGSTY YNPSLKSRVTISVDTSKNQFSLKLGSVTAA DTAVYYCARH DGTDAFDIWGQGTTVTVSS 188 Antibody EpitopeQMQLVQSGGD LVQPGGSLRL SCAASGFTFSSYSMNWVRQA 601-109 Class IVPGKGLEWVSS ISSSSSYIYY ADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARGL GGWTHDAFDIWGQGTTVTVS S 292 Antibody EpitopeQVQLVQSGGD LVQPGGSLRL SCAASGFTFSSYSMNWVRQA 601-110 Class IIPGKGLEWVSS ISSSSSYIYY ADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARGL GGWTHDAFDIWGQGTTVTVS S 293 Antibody EpitopeEVQLVQSGGD LVQPGGSLRL SCAASGFTFSSYSMNWVRQA 601-112 Class IVPGKGLEWVSS ISSSSSYIYY ADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARGL GGWTHDAFDIWGQGTTVTVS S 108 Antibody EpitopeQVQLVQSGGG LVKPGGSLRL SCAASGFTFGTYSMNWVRQA 601-119 Class IIPGKGLEWVSS ISSSSSYIYY ADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARGL GGWTHDAFDIWGQGTTVTVS S 294 Antibody EpitopeEVQLVQSGGD LVQPGGSLRL SCAASGFTFSSYSMNWVRQA 601-120 Class IVPGKGLEWVSS ISSSSSYIYY ADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARGL GGWTHDAFDIWGQGTTVTVS S 295 Antibody EpitopeEVQLVQSGAE VKKPGESLKI SCKGSGYSFSRYWIGWVRQM 601-128 Class IVPGKGLEWMGI IYPRDSDTRY SPSFQGQVTISADKSISTAYLQWSSLKASD TAMYYCATPV VTAGAFDIWGQGTMVTVSS 296 Antibody EpitopeEVQLVETGGG LVKPGGSLRL SCEASGFSLSSYSMNWVRQA 601-130 Class IPGKGLEWVSS ISSSSTHIYY ADSLKGRFTISRDNAKNSLFLQMDNLRAED TAVYYCARAT IGFDYWGQGTLVTVSS 297 Antibody EpitopeEVQLVQSGAE VKKPGASVKV SCKASGYTFTDYYIHWVRQA 601-134 Class IPGQGLEWMGW MNPNSGNSVS AQKFQGRVTMTRDTSINTAYMELSSLTSDD TAVYYCARNS EWHPWGYYDYWGQGTLVTVS S 298 Antibody EpitopeQVQLVQSGAE VKKPGASVKV SCKASGYTFSRYYIHWVRQA 601-136 Class IVPGQGLEWMGL INPGGGSTNY AQKFQGRVTMTRDTSTNTVYLELSSLRSDD TAVYYCARDY GTIDARRFDFWGQGTLVTVS S 204 Antibody EpitopeQVQLQQSGAE VKKPGASVKV SCKASGYTFSRYYIHWVRQA 601-137 Class IVPGQGLEWMGI INTDGGTTTY AQKFQGRLTMTRDTSTSTVYMELSSLRSDD TAVYYCARDY GTIDARRFDYWGQGTLVTVS S 299 Antibody EpitopeQVQLQQSGAE VKKPGASVKV SCKASGHTFSNYGISWVRQA 601 4 41 Class IIIPGQGLEWMGW ISTYNGNTNY AQKLQGRVTMTTDTSTSTAYMELRSLRSDD TAVYYCARDY YSDSSGYWDDAFDIWGQGTT VTVSS 300 Antibody EpitopeQMQLVQSGAE VKKPGASVKV SCKASGYTFSRYYIHWVRQA 601 447 Class IIIPGQGLEWMGL INPGGGSTNY AQKFQGRVTMTRDTSTNTVYLELSSLRSDD TAVYYCARDY GTIDARRFDFWGQGTLVTVS S 301 Antibody EpitopeEAQLVESGGG LVKPGGSLRL SCAASGFTFSDYYMGWVRQA 601 4 49 Class IPGKGLKWLSY ISDRAHTIYD TDSVKGRFTISRDDAKSSLYLRMNNLRVED TAVYYCARAV GAGEGFDYWGQGTLVTVSS 302 Antibody EpitopeQMQLVQSGAE VKKPGASVKV SCKASGYTFTSYYMHWVRQA 601 4 53 Class IIIPGQGLEWMGI INPSGGSTSY AQKFQGRVTMTRDTSTSTVYMELSSLRSED TAVYYCARGG YTGWSPSDPWGQGTLVTVSS

TABLE 8 Certain Light Chain CDR Sequences SEQ ID NOS: Antibody LC CDR1LC CDR2 LC CDR3   8-10 Antibody 601-1 QASQDISNYLN DASNLET QQYDNLPLT 72-74 Antibody 601-2 RASQSVSSNLA GASTRAT QQYNNWPPYT (3-12) 216-218Antibody 601-3 RASQSVSSNLA GASTRAT QQYNNWPPYT (3-16)  56-58Antibody 601-4 SGGISNVGTNGVN AMNQRPS ATWDDSLSGVL  24-26 Antibody 601-5QASQDISNYLN DASNLET QQYDNLPLT (3-2) 303-305 Antibody 601-6 QASQDIRNYLNAASNLET QQDDNLPLT 306-308 Antibody 601-9 GGTNIGSESVH DDTDRPS QVWDSVSDRYV168-170 Antibody 601-13 QASQDISNYLN DASNLET QQFDNLPYT 120-122Antibody 601-14 TGTSSDVGGYNYVS DVSNRPS SSYTSSSTLWV  88-90Antibody 601-17 RASQSVSSSYLA GASSRAT QQYGSL 309-311 Antibody 601-18RASQSVSSSYLA GASSRAT QQYGSS 312-314 Antibody 601-28 TGTSSDVGGYNYVSDVSNRPS SSYTSSSTL 315-317 Antibody 601-37 QASQDISNYLN AASSLQS QQSYSTPFT152-154 Antibody 601-40 RASQNINNYLN AASSLQS QQSYNTPFT 318-320Antibody 601-43 RASQSISSYLN AASSLQS QQSYSTPPWT 321-323 Antibody 601-50PGSSSNIGSNYVY RNNQRPS GTWDSSLSAYV  40-42 Antibody 601-51 TGTSSDFGDYDYVSDVSDRPS SSFTTSSTLV 324-326 Antibody 601-56 TGTSSDFGDYDYVS DVSDRPSSSLTTSSTLV 327-329 Antibody 601-57 RTSQSISSYLN AASSLQS QQSYSTPFT 330-332Antibody 601-65 SGGSNNIGRSSVY KTDQRPS ATWDDSLSAVV 333-335Antibody 601-66 TGTSSDFGDYDYVS DVSDRPS SSFTTSSTLV 336-338Antibody 601-69 TGTSSDVGGYNYVS DVSNRPS SSYTSSSIPWV 339-341Antibody 601-70 TGTSSDFGDYDYVS DVSDRPS SSFTTSSTLV 342-344Antibody 601-81 TGNGGRVANNYVQ EDNQRPS QSYDISNQRV 136-138 Antibody 601-86RASQGISTLLA SASSLQS QSYRAPT 345-347 Antibody 601-87 KASQDIDDDLN EATTLVPLQHDNFPPT 348-350 Antibody 601-100 TGTSRDVGGYDYVS DVSRRPS SSYTSSSTRV351-353 Antibody 601-101 RASQSISNYLN AASSLQS QQSYSTPFT 354-356Antibody 601-102 TGTSSDVGGYNYVS DVSKRPS SSYTSSSTSVV 357-359Antibody 601-103 TGTTSDVGGYNYVS DVSKRPS SSYTSSSTDV 360-362Antibody 601-108 SGSSSNIGKNYVS DNNERPS ATFDTSLWAAV 184-186Antibody 601-109 TGTSSDVGGYNYVS EVSQRPS SSYAGDRDV 363-365Antibody 601-110 TGTSSDVGGYNYVS DVTKRPS ASYTRSTTLV 366-368Antibody 601-112 TGTSSDVGGYNYVS DVSKRPS SSYTGRSTV 104-106Antibody 601-119 TGTSSDVGGYNYVT DVSKRPS SSYTSSSTLV 369-371Antibody 601-120 TGTSSDVGGYNYVS DVSNRPS SSYTSSSTRV 372-374Antibody 601-128 RASQGISSWLA AASSLQS QQANSFPLT 375-377 Antibody 601-130TGSSSNIGAGYDVH GNSNRPS QSYDSSLSGYV 378-380 Antibody 601-134 QGDSLRSYYASGKNNRPS NSRDSSGNHLV 381-383 Antibody 601-136 SGSSSNIGSNTVN SNNQRPSAAWDDSLKSFV 200-202 Antibody 601-137 SGSSSNIGSNYVY RNNQRPS AAWDDSLSAWV384-386 Antibody 601-141 RASQNSISSHLN AASSLQS QQSYSTPFT 387-389Antibody 601-147 SGSSSIGSNYVY RNNQRPS AAWDDSLSGYV 390-392Antibody 601-149 QASQDVRNYLN DATNLES QQYDNLPLS 393-395 Antibody 601-153TGTSSDVGGYNYVS DVSKRPS GSFTSSITYV

TABLE 9 Certain Heavy Chain CDR Sequences SEQ ID NOS Antibody HC CDR1HC CDR2 HC CDR3 396, 17, 18 Antibody 601- GFTFSDYYMS YISDSTNTIYYADSVKGAVGAGEGFDH 1 397, 81, 82 Antibody 601- GFSLSSFGVAVG LIYWDDDKRYSPSLKTKGGIATTGSPNWFDP 2 (3-12) 398, 225, Antibody 601- GFSLNSFGVAVGLIYWDDDRRYFPSLEG TSPMVQGIANYYAMDV 226 3 (3-16) 399, 65, 66 Antibody 601-GGSISSSNWWS EIYHSGSTNYNPSLKS DLWLGEWDL 4 400, 33, 34 Antibody 601-GFTFSDYYMG YISDRAHTIYDTDSVKG AVGAGEGFDY 5(3-2) 401-403 Antibody 601-GFTFSDYYMG YISDRAHTIYDTHSVKG AVGAGEGFDY 6 404-406 Antibody 601-GGSISRSDGYWG SIYDTGTTYYSPSLKS MGGLRSSSSDAFHT 9 407, 177, Antibody 601-GGTFSTFAIN GVIPVSGTEDYSQKFQG DRSGRDWDYFDY 178 13 408, 129, Antibody 601-GYSFTNYWLG IIYPGDSDTRYSPSFRG LNLATHTAFDI 130 14 409, 97, 98Antibody 601- GGSLSSHGVS RIIPMFGLTDYAQNFQA ESLGATFEY 17 410-412Antibody 601- GGSLSSHGVS RIIPMFGVTDYAQKFQD ESRGATFEY 18 413-415Antibody 601- GFTFSSYSMN SISSSSSYIYYADSVKG GLGGWTHDAFDI 28 416-418Antibody 601- GYTFNNYGFS WISVYNGNTNYAQKLQG DYYSDSSGYWDDAFDI 37 419, 161,Antibody 601- GYTSTNYGIS WISTYNGNTNYAQKLQG DYYSDSSGYWDDAFDI 162 40420-422 Antibody 601- GYSFGNNGITWI STYNGNTNYAQKLQG DYYSDSSGYWDDAFDI 43423-425 Antibody 601- GYTFSRYYIH LINPGGGSTNYAQKFQG DYGTIDARRFDF 50426, 49, 50 Antibody 601- GYTFTGYYMH WINPNSGGTNYAQKFQG DGDMVYDSSGPDY 51427-429 Antibody 601- GGSISSSSYYWG SIYYSGSTYYNPSLKS HDGTDAFDI 56 430-432Antibody 601- GGAFTNFGIS WISTYNSETNYAQKLQG DYYSDSSGYWDDAFDI 57 433-435Antibody 601- GDSVSSNSATWN RTYYRSKWYNDYAVSVKS GVRAFDI 65 436-438Antibody 601- GYTFRNSGIT WINPNSGGAMYVDNFQG GMADLIDVFDI 66 439-441Antibody 601- GYSFTSYWIG IIYPGDSDTRYSPSFQG LSSSSYDAFDI 69 442-444Antibody 601- GGSFKTHGIS WINPNSGGALYVDNFQG GMADLIDVFDI 70 445-447Antibody 601- GFNFDNYGLS FIYKSVNTNYSPSLKS GKVETSVVDY 81 448, 145,Antibody 601- GFSLSTRGVGVG LIYWDDDKRYSPSLKS QTMTGAFDI 146 86 449-451Antibody 601- GGSMNNYYWS RIYSSGSTNYNPALKS ASWSGTYWALFDY 87 452-454Antibody 601- GFTFSSYSMN SISSSSSYIYYADSVKG GLGGWTHDAFDI 100 455-457Antibody 601- GHSFSTYGFS WISTYNGNTNYAQKLQG DYYSDSSGYWDDAFDI 101 458-460Antibody 601- GFTFSSYSMN SISSSSSYIYYADSVKG GLGGWTHDAFDI 102 461-463Antibody 601- GFTFSSYSMN SISSSSSYIYYADSVKG GLGGWTHDAFDI 103 464-466Antibody 601- GGSISSNSYYWG SIYYSGSTYYNPSLKS HDGTDAFDI 108 467, 193,Antibody 601- GFTFSSYSMN SISSSSSYIYYADSVKG GLGGWTHDAFDI 104 109 468-470Antibody 601- GFTFSSYSMN SISSSSSYIYYADSVKG GLGGWTHDAFDI 110 471-473Antibody 601- GFTFSSYSMN SISSSSSYIYYADSVKG GLGGWTHDAFDI 112 474, 113,Antibody 601- GFTFGTYSMN SISSSSSYIYYADSVKG GLGGWTHDAFDI 114 119 475-477Antibody 601- GFTFSSYSMN SISSSSSYIYYADSVKG GLGGWTHDAFDI 120 478-480Antibody 601- GYSFSRYWIG IIYPRDSDTRYSPSFQG PVVTAGAFDI 128 481-483Antibody 601- GFSLSSYSMN SISSSSTHIYYADSLKG ATIGFDY 130 484-486Antibody 601- GYTFTDYYIH WMNPNSGNSVSAQKFQG NSEWHPWGYYDY 134 487-489Antibody 601- GYTFSRYYIH LINPGGGSTNYAQKFQG DYGTIDARRFDF 136 490, 209,Antibody 601- GYTFSRYYIH IINTDGGTTTYAQKFQG DYGTIDARRFDY 210 137 491-493Antibody 601- GHTFSNYGIS WISTYNGNTNYAQKLQG DYYSDSSGYWDDAFDI 141 494-496Antibody 601- GYTFSRYYIH LINPGGGSTNYAQKFQG DYGTIDARRFDF 147 497-499Antibody 601- GFTFSDYYMG YISDRAHTIYDTDSVKG AVGAGEGFDY 149 500-502Antibody 601- GYTFTSYYMH IINPSGGSTSYAQKFQG GGYTGWSPSDP 153

1.-44. (canceled)
 45. A polypeptide capable of binding to ReceptorTyrosine Kinase-Like Orphan Receptor 1 (ROR1) protein, the peptidecomprising an amino acid sequence selected from a group consisting of:(i) SEQ ID NO:8, 9, 10, 16, 17 and/or 18; (ii) SEQ ID NO:24, 25, 26, 32,33 and/or 34; (iii) SEQ ID NO:40, 41, 42, 48, 49 and/or 50; (iv) SEQ IDNO:56, 57, 58, 64, 65 and/or 66; (v) SEQ ID NO:72, 73, 74, 80, 81 and/or82; (vi) SEQ ID NO:88, 89, 90, 96, 97 and/or 98; (vii) SEQ ID NO:104,105, 106, 112, 113 and/or 114; (viii) SEQ ID NO:120, 121, 122, 128, 129and/or 130; (ix) SEQ ID NO:136, 137, 138, 144, 145 and/or 146; (x) SEQID NO:152, 153, 154, 160, 161 and/or 162; (xi) SEQ ID NO:168, 169, 170,176, 177 and/or 178; (xii) SEQ ID NO:184, 185, 186, 192, 193 and/or 194;(xiii) SEQ ID NO:200, 201, 202, 208, 209 and/or 210; and/or (xiv) SEQ IDNO:216, 217, 218, 224, 225 and/or
 226. 46. The polypeptide of claim 45,comprising a functional fragment of an anti-ROR1 antibody.
 47. Thepolypeptide of claim 46, comprising an anti-ROR1 scFv.
 48. Thepolypeptide of claim 45, comprising at least two, three, four, five orsix amino acid sequences defined in any of (i) to (xiv).
 49. Thepolypeptide according to claim 45, wherein the peptide comprises anamino acid sequence substantially as set out in SEQ ID NO: 4, 20, 36,52, 68, 84, 100, 116, 132, 148, 164, 180, 196 or 212, or a functionalvariant or fragment thereof, or an amino acid sequence substantially asset out in SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172,188, 204 or 220, or a functional variant or fragment thereof.
 50. Acomposition comprising a polypeptide of claim 45, optionallyderivatized, and a pharmaceutically acceptable vehicle.
 51. A nucleicacid encoding the polypeptide of claim
 45. 52. The nucleic acid of claim51, comprising a polynucleotide sequence selected from a groupconsisting of: (i) SEQ ID NO:5, 6, 7, 13, 14 and/or 15; (ii) SEQ IDNO:21, 22, 23, 29, 30 and/or 31; (iii) SEQ ID NO:37, 38, 39, 45, 46and/or 47; (iv) SEQ ID NO:53, 54, 55, 61, 62 and/or 63; (v) SEQ IDNO:69, 70, 71, 77, 78 and/or 79; (vi) SEQ ID NO:85, 86, 87, 93, 94and/or 95; (vii) SEQ ID NO:101, 102, 103, 109, 110 and/or 111; (viii)SEQ ID NO:117, 118, 119, 125, 126 and/or 127; (ix) SEQ ID NO:133, 134,135, 141, 142 and/or 143; (x) SEQ ID NO:149, 150, 151, 157, 158 and/or159; (xi) SEQ ID NO:165, 166, 167, 173, 174 and/or 175; (xii) SEQ IDNO:181, 182, 183, 189, 190 and/or 191; (xiii) SEQ ID NO:197, 198, 199,205, 206 and/or 207; and/or (xiv) SEQ ID NO:213, 214, 215, 221, 222and/or
 223. 53. The nucleic acid of claim 52, wherein the nucleic acidcomprises a nucleotide sequence substantially as set out in SEQ ID NO:3,19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195 or 211, or afunctional variant or fragment thereof, or a nucleotide sequencesubstantially as set out in SEQ ID NO:11, 27, 43, 59, 75, 91, 107, 123,139, 155, 171, 187, 203 or 219, or a functional variant or fragmentthereof.
 54. A genetic construct comprising the nucleic acid of claim50.
 55. A recombinant vector comprising the genetic construct of claim54.
 56. A host cell comprising the genetic construct of claim
 54. 57. Ahost cell comprising the polypeptide of claim
 45. 58. A compositioncomprising the nucleic acid of claim 51 and a pharmaceuticallyacceptable vehicle.
 59. A method of treating, preventing or amelioratingcancer in a subject, the method comprising administering, to a patientin need of such treatment, a therapeutically effective amount of thepolypeptide of claim 45 or the nucleic acid of claim 51, each beingoptionally derivatized.
 60. The method of claim 59, wherein thepolypeptide or nucleic acid is used in the treatment, prevention, oramelioration of a ROR1-positive cancer type.
 61. The method of claim 59,wherein the cancer is chronic lymphocytic leukemia (CLL); mantle celllymphoma (MCL); B-cell acute lymphoblastic leukemia (B-ALL); marginalzone lymphoma (MZL); neuroblastoma; renal cancer; lung cancer; or breastcancer.